M.D. Pierre Laumonerie
email: laumonerie.pierre@hotmail.fr

Laurent Blasco

Meagan E Tibbo

Kerezoudis Panagiotis

Olivier Fernandes

Frederic Lauwers

Nicolas Bonnevialle

Pierre Mansat

Xavier Ohl

Laumonerie Pierre

Blasco Laurent

Tibbo Meagan

Panagiotis Kerezoudis

Fernandes Olivier

Lauwers Frederic

Bonnevialle Nicolas

Mansat Pierre

Ohl Xavier

Ultrasound-Guided Versus Landmark-Based Approach to the Distal Suprascapular Nerve Block: A Comparative Cadaveric Study

Keywords: Conception and design, Data acquisition, Analysis and interpretation, Critical revisions, Manuscript review, Statistical analysis, Study supervision

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The suprascapular nerve (SSN) is a mixed peripheral nerve that provides the sensory and motor innervation to the posterior and superior aspects of the shoulder. Regional anesthesia obtained via SSN block (SSNb) is widely used in the management of acute, chronic, and recalcitrant shoulder pain [START_REF] Chang | Comparison of the Effectiveness of Suprascapular Nerve Block With Physical Therapy, Placebo, and Intra-Articular Injection in Management of Chronic Shoulder Pain: A Meta-Analysis of Randomized Controlled Trials[END_REF] . During the last two decades, ultrasonography (US) has gained popularity for the evaluation of peripheral neuropathies. The implementation of ultrasound-based (US) of image guidance has been utilized in an attempt to improve the accuracy of distal suprascapular nerve block (dSSNb) [START_REF] Siegenthaler | Ultrasound-guided suprascapular nerve block, description of a novel supraclavicular approach[END_REF][START_REF] Melnyk | Updated Retrospective Single-Center Comparative Analysis of Peripheral Nerve Block Complications Using Landmark Peripheral Nerve Stimulation Versus Ultrasound Guidance as a Primary Means of Nerve Localization[END_REF] . However, Lapègue et al [START_REF] Lapegue | Ultrasonography of the brachial plexus, normal appearance and practical applications[END_REF] demonstrated that the SSN was more difficult to characterize in the suprascapular location than in the supraclavicular area. The contribution of US guidance to dSSNb may be limited by the depth of the nerve, as well as the significant anatomic variation in the SS notch.

Using surface landmarks, the dSSN may also be identified at the SS notch [START_REF] Chan | Suprascapular nerve block: a narrative review[END_REF] . Landmark based approach (LBA) appears to be an attractive "low tech" alternative to the US guided approach for the dSSNb [START_REF] Matsumoto | A new nerve block procedure for the suprascapular nerve based on a cadaveric study[END_REF][START_REF] Fernandes | Suprascapular nerve block: important procedure in clinical practice. Part II[END_REF] . Despite the many available approaches and techniques (US, fluoroscopy, arthroscopy) published to date, the relative accuracy of image guidance compared to a LBA to dSSNb is unknown [START_REF] Rothe | Ultrasoundguided block of the suprascapular nerve -a volunteer study of a new proximal approach[END_REF][START_REF] Lee | Efficacy of direct arthroscopy-guided suprascapular nerve block after arthroscopic rotator cuff repair: a prospective randomized study[END_REF] . Furthermore, utilization of a LBA would allow surgeons to easily provide locoregional anesthesia to their patients in an expeditious and patient-specific fashion prior to, or subsequent to surgical procedures. The primary aim was to compare the accuracy of dSSNb performed via ultrasound-guided regional anesthesia (USRA) or a landmark-based approach (LBA). The secondary aim was to describe the anatomic features of the sensory branches of the dSSN. The primary hypothesis was that the LBA is as accurate and reliable a method as US guidance for the dSSNb. The secondary 51 hypothesis was that the dSSNb included sensory branches proximal to the SS notch.

II.

MATERIALS AND METHODS

Thirty shoulders (15 left and 15 right) from 15 fresh-frozen cadavers were obtained from the pathology department of Reims Champagne-Ardenne (Reims, France). The cadavers were stored at 4°C (median duration 5 days (range, 1-8)). The specimens were assessed by two members of the author team, an orthopedic resident and a senior anesthetist. The median age of the specimens (6 males, 9 females) was 83 years (range, 74 -98) with a median BMI of 24 kg/m 2 (range, 18.8 -30.1) [Table 1]. A history of radiation to the brachial plexus and/ or shoulder surgery, or trauma in the cervical, supraclavicular, or shoulder girdle area were considered exclusion criteria. None of the specimens were excluded. Ultrasound-guidance and LBA methods were performed in 15 (Group I) and 15 (Group II) cadaver shoulders respectively. In each cadaver, one SSN was marked using ultrasound guidance and the contralateral side was marked using the LBA. Prior to the procedure, the cadavers were thawed at room temperature for 12 hours. Anesthetic blockade was carried out within 24 hours of thawing in all cases.

GROUP I -ULTRASOUND GUIDED BLOCK OF THE DISTAL SSN

An anesthesiologist (FO) with 10 years of experience in regional anesthesia marked 15 dSSN at the SS notch using a linear high-frequency probe (10-12 MHz) (Edge II, Fujifilm Sonosite ®, Inc., Bothell, USA).

The US protocol previously described by Harmon and Hearty 9 was defined a priori and used for all 15 procedures. Marking of the dSSN was performed via a posterior approach at the level of the SS notch. Cadavers were placed in a semi-seated position at an inclination of 45º with the ipsilateral arm placed at the side of the body and the head turned to the contralateral side without neck elevation. The US probe was placed on the spine of the scapula, medial to the apophysis of the coracoid. The suprascapular notch, covered by the trapezius, was identified.

The SSN was identified beneath the superior transverse scapular ligament. Ultrasound guidance allowed us to measure the depth of the SSN at the SS notch. The needle (SonoPlex cannula 22G*80 mm (PAJUNK, Geisingen, Germany)) was inserted perpendicular to the long axis of the probe and 10 mL of 0.75% ropivacaine colored with 3 drops of methylene blue was injected around the dSSN at the SS notch in all specimens from Group 1 [Figure 1]. Subsequently, 2.5 mL of red colored latex solution was injected at the end of the 15 procedures in order to identify the position of the needle tip. The depth (distance from skin to nerve) of the SSN at the level of the injection was measured under US-guidance.

GROUPE II -LANDMARK-BASED APPROACH TO THE dSSN BLOCK

An orthopaedic resident (BL) with no experience in regional anesthesia marked 15 SSN at suprascapular notch using only bony landmarks about the shoulder.

The protocol for the LBA to dSSN blockade, previously described by Matsumoto et al [START_REF] Matsumoto | A new nerve block procedure for the suprascapular nerve based on a cadaveric study[END_REF] , was defined a priori and used for all cadavers in Group II. A block was performed using palpation alone from a posterolateral approach [START_REF] Fernandes | Suprascapular nerve block: important procedure in clinical practice. Part II[END_REF] . The operator was positioned behind the shoulder. The superficial anatomy of the shoulder was identified and the skin was marked to outline the clavicle, scapula including the acromion, and the coracoid process. The insertion point was the midpoint of the anterolateral angle of the acromion and the medial edge of the scapular spine. The needle (Hypodermic BD PrecisionGlide 21G (Becton, Dickonson and Company (BD) Franklin Lakes, NJ, US) was inclined 30° anteriorly from perpendicular in the sagittal plane and inserted until it reaches the base of the coracoid process [Figure 2]. The same injection protocol described above, including colored ropivacaine and red latex solution, was then carried out.

GROUPS I & II -DISSECTION AFTER INJECTION OF THE DISTAL SSN

To examine the effectiveness of the LBA and US guidance for the dSSNb, cadaveric dissection of the dSSN and its sensory branches was performed, in addition to dissection for anatomic description. The trapezius, deltoid, 2/3 of the medial clavicle, and spine of the scapula were removed in order to facilitate dissection into the subacromial space as well the about the rotator cuff. The supra-and infraspinatus were reflected laterally from their origins on the medial scapula. The muscles were then transected 3 cm from their insertion on the greater tuberosity of the humerus. The superior transverse scapular ligament was preserved, while the spinoglenoid ligament was reflected. Fine microsurgical instruments and surgical loops (3.5X magnification) were used for the dissection of the dSSN and its sensory branches. The mesoneurium was not preserved during the dissection.

The accuracy of the each guidance method was assessed using the proximity of the needle's position (red latex solution) to the dSSN at the SS notch, as well as to the origin of each sensory branch. Each method's predicted ability to provide reliable anesthesia was assessed via the extent of staining of the dSSN, and the origin of the most proximal sensory branch. The number of sensory branches and a description of their course, as well as the distance between the origin of the branches and the dSSN at the SS notch was also described. Distances were measured with a caliper.

STATISTICAL ANALYSIS

Continuous variables were described using medians ranges; categorical variables with frequencies and proportions. Qualitative data were compared using the Fisher-exact test, while quantitative data were compared with the Mann-Whitney-U test. A multivariable regression model was constructed to analyze the relationship between BMI and reliability (marking of the SSN at the SS notch) and accuracy (distance from SSN at the SS notch to the needle's tip) of dSSN staining; adjustments were made based on sex and age. In Group I, multivariable regression models were also used to determine the relationship between the depth of the nerve (US-measured) and the accuracy of the dSSNb; with similar adjustments. Statistical analyses were performed using Excel (Microsoft,Redmond, WA, USA) and WLSTAT 2011 (Addinsoft SARL, Paris, France) software packages. Confidence intervals (CI) were fixed at 95% and level of statistical significance was set at 0.05.

III.

RESULTS

COMPARATIVE STUDY OF METHODS FOR dSSN ANESTHETIC BLOCK

Block accuracy. The median distance between the dSSN and the site of injection was 1.4 cm (range, 0 -5.2). The injection site was distal to the SS notch in all 30 (100%) cases. Needle tip position was highly variable in both techniques; no conclusion could be drawn regarding distance from the needle tips to the SSN [Table 2]. The needle was identified at a median distance of 1.3 cm (range, 0 -5.2) and 1.5 cm (range, 0 -4.5) from the SSN with US guidance and the LBA, respectively. The higher the cadaver's BMI the lower the injection accuracy; however this correlation was not statistically significant (Coefficient (ß): 0.32; IC95 [-1,00 -1.63] ; p=0.63). The depth of the dSSN did not correlate with the distance to the needle's tip when US guidance was used (Coefficient (ß): -0.25; IC95 [-0.67-0.30], p=0.37).

Block reliability. The dSSN was stained at the level of the SS notch in 27 on 30 cadaver shoulders (90%) [Figures 2 and3]. Staining diffused past the origin of the most proximal sensory branch in all 27 cases, indicating excellent predicted block efficacy. The 3 failures that occurred in the US guidance group (80%, n = 12); also failed to mark the sensory branches with colored ropivacaine [Figure 4]. The incidence of the medial subacromial branch (MSAb), the lateral subacromial branch (LSAb), and the posterior glenohumeral branch (PGHb) was 90% (n=27), 87% (n=26), and 83% (n=25), respectively. The incidence of PGHb branch marking was significantly greater with the LBA than with US guidance (10 (67%) vs. 10 (100%), p=0.042).

The incidence of staining of the other sensory branches (MSAb and LSAb) was greater with LBA than US guidance but did not reach statistical significance (p<0.05) [Table 2]. BMI had no impact on the reliability of the anesthetic block (OR 1.04, IC95 [0.72 ; 1.49], p= 0.84.) Dispersion of the colored solution was limited to the suprascapular and SA spaces; no methylene blue was found in the infraspinous fossa. No other nervous structures were stained (eg. axillary nerve, supra-or infra-clavicular brachial plexus). There were no cases of intravascular injection.

ANATOMIC DESCRIPTION

The thirty dSNN traversed the SS notch beneath the superior transverse scapular ligament.

Three sensory branches were identified in all 30 (100%) dSSN: the medial subacromial branch (MSAb), the lateral subacromial branch (LSAb), and the posterior glenohumeral branch (PGHb) [Figures 3 and4]. Three (10%) cadaveric shoulders were found to have two LSA branches. The origin of medial and lateral SAb were at 1.2 cm (range, 0 -2.5) and 0 cm (range, 0 -1.4) from the SS notch. The majority of the origins of the MSAb were proximal to the SS notch (n=21, 70%), while the majority of LSAb originated at the level of the SS notch (n=18, 60%) with none originating proximal to it. All sensory branches that originated proximal to the SS notch, passed beneath the transverse SS ligament and through the notch. The MSAb turned supero-laterally around the base of the coracoid process, giving off branches to the coraco-clavicular ligaments (conoid and trapezoid), then ran supero-laterally toward the medial pole of the SA bursa in all cadaveric specimens. The LSAb was located deep, in the interval between the supra-and infraspinatus muscles before running over the superior face of the supraspinatus tendon and giving off sensory branches to the lateral pole of the SA space. No sensory branching to the supaspinatus tendon was found. For all 30 (100%) cadaveric shoulders, the two SA branches provided bipolar -medial and lateral -sensory innervation to the SA bursa. The origin of the PGHb was at a median distance of 3 cm distal to the SS notch; no origin of the PGHb was reported proximal to the SS notch. Distal to the spinoglenoid notch, the PGHb ran inferomedially toward the posterior shoulder capsule.

III.

DISCUSSION

The development of high-frequency US probes and high-definition US machines has made a significant impact on the efficiency of anesthetic blockade of superficial nerves (eg interscalene brachial plexus anesthetic block, femoral or sciatic nerve block) [START_REF] Hopkins | Ultrasound guidance as a gold standard in regional anaesthesia[END_REF] . However, utilizing US guidance for needle localization requires expertise and equipment availability. The impact of US on the incidence of nerve injuries remains unclear, due to the fact that the cause of transient deficits is thought to be multifactorial [START_REF] Melnyk | Updated Retrospective Single-Center Comparative Analysis of Peripheral Nerve Block Complications Using Landmark Peripheral Nerve Stimulation Versus Ultrasound Guidance as a Primary Means of Nerve Localization[END_REF] . In the present cadaveric study, we showed that the difference in the distribution of the distance of the needle to the SSN at the SS notch was not statistically significant between the two groups thereby providing evidence that the LBA technique is at least as good as the US technique.

Many techniques have been proposed to access the dSSN. They can be direct or indirect depending on whether the anesthetic solution was administered into the SS notch or into the floor of SS fossae. The LBA chosen for the present study was a direct posterior-lateral dSSNb, which was initially described and validated by Matsumoto et al [START_REF] Matsumoto | A new nerve block procedure for the suprascapular nerve based on a cadaveric study[END_REF] , which allowed for marking of 100% of the dSSN and it sensory branches via injection at a median of 1.5 cm latero-distal to the dSSN at the SS notch, irrespective of the specimens. Since its description, dSSNb has undergone several modifications including needle insertion site, and mode of access [START_REF] Fernandes | Suprascapular nerve block: important procedure in clinical practice. Part II[END_REF] . We speculate that the rate of marking of the dSSN and it sensory branches may have been lower if an indirect technique had been selected, due to the tendency to err medio-distally into the supraspinous fossa [START_REF] Chan | Suprascapular nerve block: a narrative review[END_REF] . While the LBA allowed an inexperienced orthopaedic resident to achieve excellent results, obtaining similar results with US guidance requires mastery of US and a non-trivial amount of practice [START_REF] Lapegue | Ultrasonography of the brachial plexus, normal appearance and practical applications[END_REF][START_REF] Chan | Suprascapular nerve block: a narrative review[END_REF][START_REF] Rothe | Ultrasoundguided block of the suprascapular nerve -a volunteer study of a new proximal approach[END_REF] .

Therefore the LBA is a simple and reliable block method that is easily accessible to surgeons.

Our results support the proposition that surgeons are able to "Take Back the Block" and is consistent with a thought processes detailed in "Do It Yourself" by Sherman [START_REF] Sherman | Editorial Commentary: Is It Time for Surgeons to « Take Back the Block »? Do-It-Yourself Suprascapular Nerve Blocks for Arthroscopic Rotator Cuff Repair[END_REF] .

Lapegue et al [START_REF] Lapegue | Ultrasonography of the brachial plexus, normal appearance and practical applications[END_REF] have previously demonstrated that the anatomic characteristics of the dSSN at the SS notch make US-guided procedures challenging and limits their efficiency; especially in overweight patients [START_REF] Chan | Suprascapular nerve block: a narrative review[END_REF] . To visualize the dSSN at the SS notch, the probe would have to be almost in a straight coronal position [START_REF] Melnyk | Updated Retrospective Single-Center Comparative Analysis of Peripheral Nerve Block Complications Using Landmark Peripheral Nerve Stimulation Versus Ultrasound Guidance as a Primary Means of Nerve Localization[END_REF][START_REF] Harmon | Ultrasound-guided suprascapular nerve block technique[END_REF] . However, slight deviation of the probe orientation from that position would result in scanning the floor of the suprascapular fossa. When the floor of the suprascapular fossa is scanned instead, a concave hyperechoic line appears, which can be misinterpreted as the suprascapular notch and the motor branch to the supraspinatus mistaken for the dSSN [START_REF] Harmon | Ultrasound-guided suprascapular nerve block technique[END_REF] . Needles were more likely to be placed in a medio-distal position when ultrasound guidance was used, and a lateral distal position when LBA was used. We hypothesize that the tissue plane used in the LBA (at the level of the scapular neck and base of the coracoid) allowed for better diffusion of anesthetic product than the suprascapular fossa (at the muscular insertion of the supraspinatus) used in the USRA technique. The aforementioned facts may explain the higher failure rate of the US-guided approach with respect to marking of the dSSN and its sensory branches -particularly the PGHb.

There has been significant debate in the literature with regard to the anatomic features of the sensory divisions of the SSN [START_REF] Eckmann | Cadaveric Study of the Articular Branches of the Shoulder Joint[END_REF] . We report new features of the sensory branches of the dSSN, including three branches: a medial SA branch, a lateral SA branch, and a posterior glenohumeral branch. This description is consistent with the bipolar repartition of the nocireceptors in the medial and lateral part of the subacromial bursa described by Ide et al [START_REF] Ide | Sensory nerve supply in the human subacromial bursa[END_REF] . According to Voster et al [START_REF] Vorster | The sensory branch distribution of the suprascapular nerve: an anatomic study[END_REF] , the origins of the proprioceptive branches were proximal to the SS notch in 30% of cases.

The present study found that 23.3% of the origins of all sensory branches of the dSSN arose proximal to the SS notch and consisted primarily of the MSAb (70%). The most proximal sensory branch arose 2.5 cm from the SS notch, and no branch that arose proximal to the superior transverse suprascapular ligament had a trajectory outside of the SS notch. Given the elevated rate of anatomic variation of the suprascapular notch and the possibility of passage of sensory branches outside of the SS notch, we recommend anesthetic injection and diffusion more than 2.5 cm proximal to the passage of the SSN at the SS notch. This allows for satisfactory anesthetic product diffusion following injection at the distal lateral aspect of the SS notch via LBA compared to the US method.

IV LIMITATIONS

This study is subject to the inherent biases associated with cadaveric studies. Cadaver dissection steps may have modified the normal anatomy, the needle's position, and/or the solution spreading, thereby influencing the study's finding. Furthermore, the limited number of specimens limits the reliability of descriptive parameters and limited our ability to identify statistically significant differences between the methods of dSSN block studied. We also acknowledge that US is an operator-dependent modality [START_REF] Tagliafico | Update on Ultrasound-Guided Interventional Procedures on Peripheral Nerves[END_REF] . A single, experienced anesthesiologist performed all the US characterization and regional anesthesia in this study; therefore, intra-class correlation could not be calculated. In a similar fashion, a single operator performed all blocks using the LBA. For that reason, interobserver reliability could not be determined. Additional operators would be necessary in order to better define the reliability of this technique among inexperienced surgeons. Failures of marking that occurred in the USRA group could equally have been due to operator error and not necessarily associated with the technique itself. Furthermore, the variation in accuracy and reliability of USRA according to operator experience and the lack of standardized dye dispersion (ie, needle size, rate of infusion) could not be assessed. The volume of solution injected was based on Wiegel et al [START_REF] Wetzel | Interventions for Postsurgical Opioid Prescribing: A Systematic Review[END_REF] and Desroches et al's [START_REF] Desroches | Suprascapular Nerve Block Versus Interscalene Block as Analgesia After Arthroscopic Rotator Cuff Repair: A Randomized Controlled Noninferiority Trial[END_REF] recommendations; however, excessive dispersion of the staining dye increases the chance that the dSSN will be marked. A single, experienced anesthesiologist performed all the US characterization and regional anesthesia in this study; therefore, intra-class correlation could not be calculated. The lack of visible blood flow during US in addition to the fact that we did not utilize "needle enhancement" mode (Sonosite Edge II) made it more challenging to describe the dSSN at the suprascapular notch. Pleuro-pulmonary injuries, systemic toxicity, and peripheral nerve injury are well diescribed complications of SSN anesthetic block; however, these were not evaluated in the present study. Melnyk et al [START_REF] Melnyk | Updated Retrospective Single-Center Comparative Analysis of Peripheral Nerve Block Complications Using Landmark Peripheral Nerve Stimulation Versus Ultrasound Guidance as a Primary Means of Nerve Localization[END_REF] showed that while the use of guidance during peripheral nerve blocks leads to a significant reduction in the incidence of local anesthetic systemic toxicity, the frequency of lasting injuries did not differ significantly. In addition, the impact of US on the incidence of nerve injuries remains unclear, due to the fact that the cause of transient deficits is thought to be multifactorial. Finally, the lack of statistical significance might also be attributed to the relatively small sample size. Ad hoc power analysis revealed that with 15 cadaveric shoulders per arm (30 total), we would have 75% power to detect a clinically significant difference of 10 mm in the needle distance to the SSN at the SS notch between the LBA and the US group (effect size of 1), assuming a 2-sided significance level of 0.05. Nevertheless, the number of cadavers used in this study is similar to that employed in most contemporary relevant orthopedic studies [START_REF] Matsumoto | A new nerve block procedure for the suprascapular nerve based on a cadaveric study[END_REF][START_REF] Rothe | Ultrasoundguided block of the suprascapular nerve -a volunteer study of a new proximal approach[END_REF][START_REF] Lee | Efficacy of direct arthroscopy-guided suprascapular nerve block after arthroscopic rotator cuff repair: a prospective randomized study[END_REF] .

IV.

CONCLUSION

A LBA is as reliable and accurate as US guidance for the dSSN anesthetic block. Marking of the SSN must be proximal to the SS notch in order to involve the 3 sensory branches in the anesthetic blockade. Only the branch to the supraspinatus (SSp) was marked.

TABLES AND FIGURES

Figure 1 .

 1 Figure 1. Ultrasonographic of the suprascapular nerve in a left shoulder. (A) A linear high

Figure 2 .Figure 3 .

 23 Figure 2. Identification of the dSSN via the LBA in a right shoulder. (A) The LBA consisted

Figure 4 .

 4 Figure 4. Failed Marking of the dSSN at the SS notch via US guidance in a left shoulder.

Table 1 .

 1 Characteristics of the study group: ultrasound guided and landmark-based approach to the distal suprascapular nerve block

Table 2 .

 2 Results of marking the dSSN at the SS notch with US guidance or LBA in cadavers.

Table 1 .

 1 A, acromion; C, Clavicle; CCI, Coracoclavicular ligaments; ISp, Branches to Supra and infraspinatus muscles; L and MSAb medial and lateral subacromial branches; PGHP, Posterior gleno-humeral branch; SA or *, subsubacromial bursa; SE supraepinous muscle Characteristtics of the study cohort : ultrasoud guided and landmarks based approach of

	suprascapular nerve block
	*	Skin -SSN distance measured with ultrasound
	N/A Not avalaible
			US guided	Bony landmark-based	P value
			n= 15	n= 15
	Sex				1
	• Males n (%)	6 (50%)	6 (50%)
	• Female, n (%)	9 (50%)	9 (50%)
	Age, median	84	82	0,754
	BMI (Kg/m 2 )	22,7	24	0,371
	Side				1
	• Left, n (%)	7 (50%)	7 (50%)
	• Right, n (%)	8 (50%)	8 (50%)
	Depth SSN*, median (mm)	21	N/ A

TABLE 2 399 Results of marking of the dSSN at the SS notch with US guidance or LBA in cadavers

 2

	400
	401
	402
	403
	404
	405

This research received no specific grant from any funding agency in the public, commercial, or not for profit sectors.

US guided


Geoffrey Terral

Arnau Busquets-Garcia

Marjorie Varilh

Svein Achicallende

Astrid Cannich

Itziar Luigi Bellocchio

Bonilla-Del Río

Federico Massa

Nagore Puente

Edgar Soria-Gomez

Itziar Bonilla

-Del Río

Pedro Grandes

Guillaume Ferreira
email: guillaume.ferreira@inra.fr

Giovanni Marsicano
email: giovanni.marsicano@inserm.fr

CB1 Receptors in the anterior piriform cortex control odor preference memory

Keywords: anterior piriform cortex, CB1 receptors, conditioned odor preference, conditioned odor aversion, neuroanatomy, miniature inhibitory currents (mIPSCs), semilunar neurons, pyramidal neurons

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Chemosensory information is crucial for the survival of humans and other animals. For example, a large part of animal behavior including control of emotional states, food intake and social interactions relies on the capacity to perceive odor information and to retrieve its potential meaning based on previous experiences [START_REF] Smeets | Smelly primes -when olfactory primes do or do not work[END_REF][START_REF] Sullivan | Olfactory memory networks: from emotional learning to social behaviors[END_REF].

Olfactory perception starts when odorant molecules, travelling through orthonasal or retronasal pathways, reach olfactory receptors on sensory neurons located in the olfactory epithelium [START_REF] Buck | A Novel Multigene Family May Encode Odorant Receptors: A Molecular Basis for Odor Recognition[END_REF]. These neurons project to the olfactory bulb that in turn transmits the signal to other brain regions, including the anterior piriform cortex (aPC) [START_REF] Ghosh | Sensory maps in the olfactory cortex defined by long-range viral tracing of single neurons[END_REF][START_REF] Sosulski | Distinct representations of olfactory information in different cortical centres[END_REF], which plays a key role in olfactory processing and memory [START_REF] Bekkers | Neurons and circuits for odor processing in the piriform cortex[END_REF][START_REF] Neville | Olfactory cortex[END_REF][START_REF] Wilson | Cortical Processing of Odor Objects[END_REF]. However, the specific cellular mechanisms governing odor information storage and retrieval in the aPC are still unclear.

Cannabinoid type-1 (CB1) receptors together with their endogenous ligands (endocannabinoids) form the core of the so-called endocannabinoid system (ECS) in the brain [START_REF] Piomelli | The molecular logic of endocannabinoid signalling[END_REF], which is an important modulator of many functions, including learning and memory [START_REF] Drumond | Endocannabinoid signaling and memory dynamics: A synaptic perspective[END_REF][START_REF] Marsicano | Roles of the endocannabinoid system in learning and memory[END_REF]. Activation of presynaptic CB1 receptors is well-known to physiologically control the release of several neurotransmitters in many brain regions [START_REF] Araque | Synaptic functions of endocannabinoid signaling in health and disease[END_REF][START_REF] Kano | Endocannabinoid-Mediated Control of Synaptic Transmission[END_REF]. CB1 receptors are present in different olfactory structures [START_REF] Marsicano | Anatomical Distribution of Receptors, Ligands and Enzymes in the Brain and in the Spinal Cord: Circuitries and Neurochemistry[END_REF], where they can modulate olfactory processes [START_REF] Ghosh | Learning-induced modulation of the effect of endocannabinoids on inhibitory synaptic transmission[END_REF][START_REF] Pouille | Cannabinoid Receptors Modulate Excitation of an Olfactory Bulb Local Circuit by Cortical Feedback[END_REF][START_REF] Soria-Gómez | New insights on food intake control by olfactory processes: The emerging role of the endocannabinoid system[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] Wang | Cannabinoid receptor-mediated regulation of neuronal activity and signaling in glomeruli of the main olfactory bulb[END_REF]. However, little is known about the specific impact of CB1 receptor signaling in olfactory brain structures on odor-dependent memory functions.

Considering that the aPC is an important region for olfactory memory [START_REF] Bekkers | Neurons and circuits for odor processing in the piriform cortex[END_REF][START_REF] Neville | Olfactory cortex[END_REF][START_REF] Wilson | Cortical Processing of Odor Objects[END_REF][START_REF] Barnes | Olfactory perceptual stability and discrimination[END_REF][START_REF] Mediavilla | Role of anterior piriform cortex in the acquisition of conditioned flavour preference[END_REF][START_REF] Schoenbaum | Information coding in the rodent prefrontal cortex. I. Single-neuron activity in orbitofrontal cortex compared with that in pyriform cortex[END_REF], we hypothesized that CB1 receptors in the aPC (aPC-CB1) could modulate odorrelated memory processes. Our data show that aPC-CB1 receptors are specifically required for the expression of appetitive, but not aversive, olfactory memory, and they are involved in the direct control of the associated modulation of local inhibitory circuits. Altogether, these results indicate that the physiological activation of CB1 receptors in the aPC exerts a fine-tuned regulation of olfactory circuits and functionally discriminates the retrieval of positively-and negatively-motivated olfactory memories.

RESULTS

CB1 receptors in the aPC are necessary for the retrieval of conditioned odor preference

To investigate the potential impact of CB1 receptor signaling in odor-related memory, we set up a behavioral protocol to assess conditioned odor preference (COP) in mice (see STAR Methods, Figures 1A andS1A,B) [START_REF] Rusiniak | Flavor-illness aversions: potentiation of odor by taste in rats[END_REF][START_REF] Slotnick | Detection and discrimination of propionic acid after removal of its 2-DG identified major focus in the olfactory bulb: a psychophysical analysis[END_REF]. Using this protocol, mice displayed a reliable preference for the odor-scented solution previously associated to sucrose (C+) as compared to the other one (C-), revealing the formation of COP (Figure S1C), regardless of the odor used as C+ (Figures S1D,E).

We next investigated the role of CB1 receptors in the aPC (aPC-CB1) during COP. Specific deletion of the CB1 gene in the aPC (aPC-CB1-KO, see STAR Methods, Figures 1B,C andS1F-I) [START_REF] Busquets-Garcia | Hippocampal CB1 Receptors Control Incidental Associations[END_REF][START_REF] Marsicano | CB1 cannabinoid receptors and on-demand defense against excitotoxicity[END_REF][START_REF] Monory | The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus[END_REF] abolished the preference for the C+ solution during test (Figure 1D), without altering sucrose preference upon training (Figure S1J), nor total liquid intake (Figure S1K), and independently from the anatomical extension of the CB1 deletion (Figure S1L). These results indicate that COP requires aPC-CB1 receptors. To determine the specific role of CB1 receptor signaling in the different phases of the COP protocol, we acutely injected the CB1 receptor antagonist AM251 into the aPC (4µg/0.5µl per side; Figures S2A-E) prior to each odor-sucrose pairing or before the retrieval test (Figure 1A). Neither consumption during training, nor COP performance were affected by aPC-CB1 blockade before each pairing (AM pairings; Figures 1D, S1J,K and S2F). Conversely, AM251 acutely injected into the aPC prior to the retrieval test abolished COP, without altering total liquid consumption (AM test; Figures 1D, S1J,K and S2F). Acute blockade of aPC-CB1 receptors might permanently impair COP retrieval. On the other hand, longer training might render COP retrieval independent of aPC-CB1 receptors. To simultaneously test for these possibilities, animals previously treated with AM251 or vehicle received 4 additional odor-sucrose pairings (see STAR Methods) and were injected with vehicle ["Veh(AM)

test"] or AM251 ["AM(Veh) test"] before the second retrieval test, respectively. In these conditions, vehicle-treated mice displayed clear COP, whereas AM251 blocked this behavior (Figures 1E andS2G). aPC-CB1 receptors might control expression of preference independently of previous learning. However, aPC-CB1 blockade did not impair innate sucrose preference (Figures S3A-C).

Altogether, these results indicate that endogenous activation of aPC-CB1 is specifically required during retrieval of COP, without affecting innate responses to attractive stimuli.

CB1 receptors in the aPC are not involved in the retrieval of conditioned odor aversion

We next asked whether aPC-CB1 receptors are also involved in the retrieval of conditioned odor aversion (COA) induced by lithium chloride (LiCl) injections (Figure 2A, COA-lithium, COA-L) [START_REF] Busquets-Garcia | Hippocampal CB1 Receptors Control Incidental Associations[END_REF][START_REF] Busquets-Garcia | Representation-mediated Aversion as a Model to Study Psychotic-like States in Mice[END_REF][START_REF] Busquets-Garcia | Pregnenolone blocks cannabinoid-induced acute psychotic-like states in mice[END_REF][START_REF] Soria-Gómez | Habenular CB1 Receptors Control the Expression of Aversive Memories[END_REF]. Notably, AM251 acutely injected into the aPC did not impair COA-L expression (Figures 2A,B, S2A-F and S3D), suggesting that aPC-CB1 receptors are dispensable for the retrieval of negatively motivated olfactory memory. However, the differential effects of CB1 receptor blockade between COP and COA-L could be due to the different types of associations involved (sensorysensory versus sensory-gastric). Using a sensory-sensory COA protocol, where sucrose was substituted by the aversive taste quinine (COA-Q; Figures 2C andS3E) [START_REF] Sevelinges | The basolateral amygdala is necessary for the encoding and the expression of odor memory[END_REF][START_REF] Yasoshima | Different disruptive effects on the acquisition and expression of conditioned taste aversion by blockades of amygdalar ionotropic and metabotropic glutamatergic receptor subtypes in rats[END_REF], mice treated with vehicle or the CB1 receptor antagonist before the retrieval test displayed the same avoidance towards the conditioned odor (Figures 2D, S2F Altogether, these data indicate that aPC-CB1 receptor signaling is necessary for the retrieval of COP but not of COA, suggesting that expression of acquired odor choices rely on different mechanisms depending on the valence of the unconditioned stimulus.

CB1 receptors are highly expressed in GABAergic interneurons in the aPC

As previously reported [START_REF] Marsicano | Anatomical Distribution of Receptors, Ligands and Enzymes in the Brain and in the Spinal Cord: Circuitries and Neurochemistry[END_REF][START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid CB1 receptor in cell-type-specific mutant mouse rescue models[END_REF], fluorescent immunohistochemistry revealed that CB1 receptor protein is highly expressed in layer II of the aPC (Figure 3A), where the aPC principal neurons are mainly localized [START_REF] Suzuki | Neural Coding by Two Classes of Principal Cells in the Mouse Piriform Cortex[END_REF][START_REF] Suzuki | Two Layers of Synaptic Processing by Principal Neurons in Piriform Cortex[END_REF]. To detail the cellular distribution of CB1 receptors, we analyzed aPC tissues from conditional mutant mice carrying exclusive expression of the protein in identified specific cell types (Rescue mice) [START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid CB1 receptor in cell-type-specific mutant mouse rescue models[END_REF][START_REF] Remmers | Addressing sufficiency of the CB1 receptor for endocannabinoid-mediated functions through conditional genetic rescue in forebrain GABAergic neurons[END_REF][START_REF] Ruehle | Cannabinoid CB1 Receptor in Dorsal Telencephalic Glutamatergic Neurons: Distinctive Sufficiency for Hippocampus-Dependent and Amygdala-Dependent Synaptic and Behavioral Functions[END_REF]. A similar pattern of CB1 receptor immunoreactivity was observed across aPC tissues from WT mice and global CB1-Rescue mice (Figure 3A) [START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid CB1 receptor in cell-type-specific mutant mouse rescue models[END_REF][START_REF] Ruehle | Cannabinoid CB1 Receptor in Dorsal Telencephalic Glutamatergic Neurons: Distinctive Sufficiency for Hippocampus-Dependent and Amygdala-Dependent Synaptic and Behavioral Functions[END_REF], but also from mice with specific re-expression in GABAergic neurons (GABA-CB1 Rescue; Figure 3A) [START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid CB1 receptor in cell-type-specific mutant mouse rescue models[END_REF][START_REF] Remmers | Addressing sufficiency of the CB1 receptor for endocannabinoid-mediated functions through conditional genetic rescue in forebrain GABAergic neurons[END_REF]. In contrast, the immunoreactivity was extremely low in mice reexpressing the receptor only in cortical glutamatergic neurons (Glu-CB1 Rescue; Figure 3A) [START_REF] Ruehle | Cannabinoid CB1 Receptor in Dorsal Telencephalic Glutamatergic Neurons: Distinctive Sufficiency for Hippocampus-Dependent and Amygdala-Dependent Synaptic and Behavioral Functions[END_REF] and, as expected, was undetectable in mice where CB1 receptor expression is absent (CB1 Stop; Figure 3A) [START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid CB1 receptor in cell-type-specific mutant mouse rescue models[END_REF][START_REF] Remmers | Addressing sufficiency of the CB1 receptor for endocannabinoid-mediated functions through conditional genetic rescue in forebrain GABAergic neurons[END_REF][START_REF] Ruehle | Cannabinoid CB1 Receptor in Dorsal Telencephalic Glutamatergic Neurons: Distinctive Sufficiency for Hippocampus-Dependent and Amygdala-Dependent Synaptic and Behavioral Functions[END_REF]. To better characterize the expression of CB1 receptors at the synaptic level, we next used immunogold electron microscopy (Figure 3B). As expected, CB1 receptor immunogold particles were specifically present in different amounts at many cellular locations (Figures 3B andS4). Amongst CB1 receptor particles located at terminals (1856 over 3409 total counted in WT; Figures 3B andS4), approximately 88% and 12% were at symmetric (presumably inhibitory) and asymmetric synapses (presumably excitatory), respectively (Figures 3B,C). Moreover, whereas only 23% of excitatory terminals were labeled with CB1 receptor immunogold particles, this percentage was as high as 87% in inhibitory ones (Figures 3B,D). As expected, only background staining was detected in sections from CB1-KO mice (18 particles at terminals; Figures 3B,D andS4).

To identify the specific topographical distribution of CB1-expressing cells within the aPC, we used double Fluorescent In Situ Hybridization (D-FISH) to label the mRNAs of CB1 receptor and of glutamic acid decarboxylase 65KDa (GAD), a marker of GABAergic neurons (Figure 4A). Consistent with previous studies [START_REF] Suzuki | Inhibitory neurons in the anterior piriform cortex of the mouse: Classification using molecular markers[END_REF], the majority of GAD-expressing cells were observed in deep layer III (Figures 4A,B). CB1 mRNA was also highly expressed in layer III, with scattered positive cells in layer I (Figures 4A,C). Accordingly, counting of positive cells revealed that a high proportion of GAD+ neurons contained also CB1 mRNA (63%; Figures 4A,D), following the distribution of GAD across layers (Figures 4A,B,E). Similarly to other cortical regions such as the hippocampus [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF], CB1 mRNA was expressed at very different levels across CB1positive aPC cells. Whereas a majority of cells expressed low-to-moderate amounts of the transcript, scattered cells contained very high levels of CB1 mRNA, especially in layers II and III (Figure 4A). Interestingly, virtually 100% of these high CB1expressing neurons co-expressed GAD mRNA (Figure 4F). Conversely, low CB1expressing neurons were virtually all identified as GAD-positive in layer I, but this proportion was reduced in layers II and III (Figure 4F).

Altogether, these data indicate that the large majority of aPC-CB1 receptor protein is expressed in terminals of local GABAergic neurons, with layer-specific topographical distribution.

CB1 receptors control inhibitory transmission in the aPC

To start addressing the so-far unexplored cannabinoid-dependent control of inhibitory transmission in the aPC, we first determined by ex vivo patch-clamp electrophysiological recordings the impact of CB1 receptor activation on GABAergic neurotransmission impinging onto specific populations of principal cells of the aPC (semilunar-like neurons, SL, and pyramidal-like neurons, PNs; Figures S5A-B and see STAR Methods). These cells receive many inputs from the olfactory bulb and other brain regions, project to other olfactory cortical areas [START_REF] Suzuki | Two Layers of Synaptic Processing by Principal Neurons in Piriform Cortex[END_REF][START_REF] Mazo | Distinct projection patterns of different classes of layer 2 principal neurons in the olfactory cortex[END_REF][START_REF] Haberly | Association and commissural fiber systems of the olfactory cortex of the rat. I. Systems originating in the piriform cortex and adjacent areas[END_REF] and are extensively innervated by local inhibitory interneurons [START_REF] Suzuki | Inhibitory neurons in the anterior piriform cortex of the mouse: Classification using molecular markers[END_REF][START_REF] Suzuki | Microcircuits Mediating Feedforward and Feedback Synaptic Inhibition in the Piriform Cortex[END_REF][START_REF] Poo | Odor Representations in Olfactory Cortex: "Sparse" Coding, Global Inhibition, and Oscillations[END_REF]. Thus, we reasoned that selective modulation of GABAergic inputs onto SL cells and/or PNs might represent a way through which CB1 receptors rapidly regulate principal cell activity and consequently odor processing. To address this possibility, we recorded miniature inhibitory post-synaptic currents (mIPSCs) representing the global inhibitory inputs of principal cells. These events occur at a frequency of 2.06 ± 0.23 Hz and an amplitude of 76.29 ± 4.41 pA (Vehicle; Figures 5A-C) in SL cells, and at a frequency of 2.50 ± 0.39 Hz and an amplitude of 64.06 ± 4.27 pA in PNs of naïve animals (Vehicle; Figures 5D-F). Similarly to what observed in the hippocampus [START_REF] Wilson | Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses[END_REF], the application of the CB1 receptor agonist WIN 55,212-2 (WIN, 5µM) significantly reduced the frequency of mIPSCs in SL cells, with only a slight impact, if any, on their amplitude (Figures 5A-C), suggesting a presynaptic inhibitory effect. This decrease was fully reversed by the application of the CB1 receptor antagonist AM251 (4µM; WIN+AM251; Figures 5A-C). Conversely and surprisingly, no such effects were observed in PNs (Figures 5D-F), indicating a cell-type specific impact of CB1 receptor-dependent control of inhibitory currents in naïve animals.

In summary, CB1 receptors are highly expressed in GABAergic interneurons of the aPC, and their activation results in the modulation of inhibitory inputs onto SL principal neurons.

COP retrieval reduces mIPSCs in the aPC via presynaptic CB1 receptors

We next hypothesized that retrieval of COP might be associated with aPC-CB1 receptor-dependent modulation of principal cells' mIPSCs. A significant reduction of mIPSCs frequency (~26%) was found in SL cells from mice sacrificed during COP retrieval, as compared to a control group exposed to the same number of only waterdrinking sessions (Water; Figures 6A,B). SL cells from mice receiving the same number of "training" sessions, but without the presence of sucrose (Sucrose free) or odor (Odor free) did not display any reduction of mIPSCs frequency (Figure 6B).

Considering that aPC-CB1 receptors regulate COP but not COA retrieval (Figure 1 and Figure 2), we next evaluated the effect of COA-Q retrieval and we found that this condition did not affect mIPSCs frequency (Figure 6B). Notably, no differences in amplitudes were observed across the groups (Figure S6A). Altogether, these results indicate that COP retrieval, but not exposure to odor or sucrose alone or COA retrieval, is associated with a specific reduction of presynaptic inhibitory transmission onto SL neurons in the aPC.

As local blockade, the systemic injection of the CB1 receptor antagonist Rimonabant (Rim, 1 mg/kg) impaired COP retrieval (Figures 6C andS6B), independently of total liquid consumption (Figures S6C-F). As expected, systemic injection of vehicle before COP retrieval did not alter the associated reduction of mIPSCs frequency in SL cells (Figures 6D,E; p>0.8, as compared to Figure 6B). Conversely, systemic administration of Rim abolished this decrease up to levels undistinguishable from control mice (Figures 6D,E; p>0.8, as compared to Figure 6B), with no effect on amplitude (Figure S6G). Notably, mIPSC frequencies, but not amplitudes, of individual animals were inversely correlated with the COP retrieval performance (Figure 6F and data not shown), suggesting that the level of presynaptic inhibition of SL cells is linked to the behavioral retrieval of COP. Next, we examined the impact of COP retrieval on mIPSCs of PNs.

Similarly to SL neurons, the frequency of mIPSCs was reduced (~33%) in PNs of animals undergoing COP retrieval, as compared to water control mice (Figures 6G-H) with no change in amplitude (Figure S6H). A slight non-significant increase of mIPSCs amplitude was observed in PNs from mice receiving systemic injection of Rim prior COP retrieval (Figure S6H). However, this treatment was not able to reverse the COP retrieval-associated reduction of mIPSCs frequency in PNs (Figures 6G-H). No correlations between COP retrieval and the levels of mIPSC frequencies or amplitudes of PNs in individual mice were observed (Figure 6I and data not shown).

Altogether, these results indicate that COP retrieval is associated to a reduction of inhibitory inputs on both SL cells and PNs. However, presynaptic CB1 receptors appear to regulate inhibitory transmission in the aPC in a cell type-specific manner, thereby providing an unforeseen fine-tuned modulation of olfactory memory circuits, likely contributing to appropriate behavioral responses.

Acute blockade of CB1 receptors affects COP retrieval through GABAergic neurons

We then addressed the potential involvement of CB1 receptors expressed in inhibitory neurons (GABAergic CB1) in COP retrieval. Surprisingly, mice lacking CB1 gene expression from GABAergic neurons (GABA-CB1-KO) [START_REF] Monory | The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus[END_REF][START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF] did not display any alteration of COP retrieval as compared to control wild-type littermates (GABA-CB1-WT; Figure S6I). This negative result might suggest that CB1 receptors in GABAergic neurons are not necessary for acute COP retrieval. However, GABA-CB1-KO mice carry a deletion of CB1 receptors in all GABAergic cells of the whole forebrain, starting from early developmental stages [START_REF] Monory | The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus[END_REF][START_REF] Zerucha | A Highly Conserved Enhancer in the Dlx5/Dlx6 Intergenic Region is the Site of Cross-Regulatory Interactions between Dlx Genes in the Embryonic Forebrain[END_REF]. Such diffuse and longlasting absence of CB1 receptor signaling might induce opposing effects in different brain regions and/or stimulate developmental compensatory phenomena, whose general mechanisms recently started to be elucidated [START_REF] El-Brolosy | Genetic compensation: A phenomenon in search of mechanisms[END_REF][START_REF] El-Brolosy | Genetic compensation triggered by mutant mRNA degradation[END_REF]. If occurring in GABA-CB1-KO mice, these mechanisms might mask the specific acute role of CB1 receptors signaling in GABAergic neurons in COP retrieval during adulthood.

Considering this possibility, we adopted an alternative strategy to investigate the specific role of CB1 receptors in GABAergic neurons, by testing whether the blockade of COP retrieval by the acute administration of Rim was still effective in GABA-CB1-KO mice. A systemic acute injection of the drug blocked COP retrieval in GABA-CB1-WT, but it failed to impair this behavior in GABA-CB1-KO mice (Figures 6J,K and S6J-L), indicating that CB1 receptors in GABAergic neurons are required for the COP retrieval-disrupting effect of the acute pharmacological blockade of CB1 receptor signaling.

DISCUSSION

In this study, we functionally characterized the presence and the role of CB1 receptors in the aPC. We found that these receptors are specifically involved in the retrieval of appetitive, but not aversive, olfactory memory and in the associated modulation of inhibitory transmission onto specific aPC principal cells. Moreover, our data show that the retrieval impairment of appetitive olfactory memory induced by CB1 antagonism requires CB1 receptors expressed in inhibitory neurons.

aPC-CB1 receptors are necessary for the retrieval of appetitive but not aversive olfactory memory

In other brain structures, CB1 receptors have been reported to play crucial roles in different phases of learning and memory processes [START_REF] Drumond | Endocannabinoid signaling and memory dynamics: A synaptic perspective[END_REF][START_REF] Marsicano | Roles of the endocannabinoid system in learning and memory[END_REF]. Our data reveal that the endogenous activation of aPC-CB1 receptors is necessary for COP retrieval, but it is dispensable for its acquisition, thereby enlarging the spectrum of CB1 receptor involvement in different phases of learning and memory. Interestingly, it was recently found that a small enhancement of hippocampal GABAergic inhibition blocked cell firing and memory retrieval but left memory encoding intact [START_REF] Rossato | Silent Learning[END_REF]. This nicely parallels our results showing that a small, but CB1 receptor-and cell type-dependent reduction in GABAergic inhibition in the aPC, is associated with effective COP memory retrieval. We here propose that CB1 receptors on GABAergic axon terminals might be essential to reduce inhibition onto SL cells, thereby permitting cell firing in the ensemble population to retrieve the COP memory trace. In this context, it is interesting to note that COP retrieval is also associated with a similar reduction of inhibitory drive onto PNs, but in a CB1 receptor-independent manner. This suggests that cell type-specific mechanisms are involved in the coordinated regulation of the activity of distinct aPC principal neurons during retrieval of olfactory memory.

Our data show that the retrieval of aversive olfactory memory is independent of aPC-CB1 receptor signaling. These intriguing results might be explained by two possibilities: either COA depends on aPC but it does not involve CB1 receptor signaling, or COA does not depend on aPC. There is currently no clear answer to this question, but some pieces of evidence seem to indicate a certain level of specialization of the aPC for positively motivated olfactory memory. Optogenetic or chemogenetic manipulations of selected neurons in the PC can modulate both aversive and appetitive behavioral responses [START_REF] Choi | Driving Opposing Behaviors with Ensembles of Piriform Neurons[END_REF][START_REF] Meissner-Bernard | Encoding of Odor Fear Memories in the Mouse Olfactory Cortex[END_REF]. Despite the fact that specific aPC activity was observed during COA retrieval [START_REF] Chapuis | The Way an Odor Is Experienced during Aversive Conditioning Determines the Extent of the Network Recruited during Retrieval: A Multisite Electrophysiological Study in Rats[END_REF], olfactory cues associated with sucrose activate more aPC neurons than odors associated with quinine [START_REF] Roesch | Associative Encoding in Anterior Piriform Cortex versus Orbitofrontal Cortex during Odor Discrimination and Reversal Learning[END_REF] and aPC lesions impair appetitive but not aversive odor-related memory [START_REF] Mediavilla | Role of anterior piriform cortex in the acquisition of conditioned flavour preference[END_REF]. In addition, CB1 receptor signaling can mediate aversive olfactory memory in other brain regions. For instance, [START_REF] Laviolette | Cannabinoids Potentiate Emotional Learning Plasticity in Neurons of the Medial Prefrontal Cortex through Basolateral Amygdala Inputs[END_REF] showed that CB1 receptors in the medial prefrontal cortex are required for odor-dependent fear conditioning [START_REF] Laviolette | Cannabinoids Potentiate Emotional Learning Plasticity in Neurons of the Medial Prefrontal Cortex through Basolateral Amygdala Inputs[END_REF], and we recently demonstrated that deletion of the CB1 gene specifically in medial habenular neurons selectively abolishes COA, but not COP [START_REF] Soria-Gómez | Habenular CB1 Receptors Control the Expression of Aversive Memories[END_REF]. Moreover, it has been shown that the basolateral nucleus of the amygdala (BLA), which is essential for COA [START_REF] Sevelinges | The basolateral amygdala is necessary for the encoding and the expression of odor memory[END_REF][START_REF] Miranda | Basolateral amygdala noradrenergic activity is involved in the acquisition of conditioned odor aversion in the rat[END_REF], is more strongly connected with the posterior PC (pPC) than with the aPC [START_REF] Luna | Input-specific excitation of olfactory cortex microcircuits[END_REF], and BLA-pPC interactions are important for aversive odor conditioning [START_REF] Hegoburu | Differential involvement of amygdala and cortical NMDA receptors activation upon encoding in odor fear memory[END_REF].

Conversely, the aPC is more densely connected than the pPC to the olfactory tubercle, which, by receiving intense dopaminergic inputs, might be specifically involved in the processing of reward-related information [START_REF] Wesson | Sniffing out the contributions of the olfactory tubercle to the sense of smell: Hedonics, sensory integration, and more?[END_REF]. Altogether, this suggests a potential double dissociation in the roles of aPC and pPC in COP and COA, with the aPC being somehow specialized in processing positive acquired values of odors and the pPC more involved in aversive odor memory.

CB1 receptors are highly expressed in aPC GABAergic interneurons and regulate local inhibitory neurotransmission

Our immunohistochemical, D-FISH and electron microscopy data show that CB1 receptors are found in a high proportion of GABAergic neurons located in the three layers of the aPC and that they are strongly expressed at inhibitory synaptic terminals. Moreover, pharmacological activation of aPC-CB1 receptors decreases miniature inhibitory currents frequency specifically in SL cells. Similarly to the hippocampus [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF], cells expressing high levels of CB1 mRNA are exclusively GABAergic interneurons. Recent evidence points to the presence of long-range inhibitory neurons as a novel neuroanatomical and functional feature in cortical areas [START_REF] Caputi | The long and short of GABAergic neurons[END_REF]. These putative long-range inputs to the aPC might contain CB1 receptors, but our viral manipulations exclude their participation in the CB1 receptor-dependent retrieval of COP. Interestingly, depending on the layer, a portion of low CB1expressing cells do not co-express GAD mRNA and are presumably glutamatergic neurons. Indeed, CB1 receptor protein is abundantly present in the main olfactory bulb at terminals of glutamatergic centrifugal fibers coming from principal neurons of the anterior olfactory nucleus and aPC [START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF]. Importantly, as these "glutamatergic" CB1 receptors play a key role in the control of olfactory perception and food intake [START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF], we cannot fully exclude that alterations in olfactory perception might participate in the phenotype of aPC-CB1-KO mice. However, intra-aPC pharmacological manipulations indicate that local CB1 receptor signaling is necessary for COP retrieval, but it is dispensable for expression of COA. Therefore, any putative impairment of olfactory perception induced by deletion of the CB1 gene in projecting glutamatergic neurons of the aPC is unlikely to be responsible for the phenotype of aPC-CB1-KO mice. Nevertheless, our electron microscopy immunogold results indicate that a small proportion of CB1 receptors are specifically present at glutamatergic terminals within the aPC. Moreover, a consistent portion of CB1 receptors appears to be located outside of terminals. Future studies will investigate the origin and the potential roles of these aPC subpopulations of CB1 receptors, which might still have functional significance.

COP retrieval is associated with CB1 receptor-dependent modulation of inhibitory transmission on specific aPC principal cells

An approximate 30% decrease of mIPSCs frequency recorded in both aPC SL cells and PNs was observed in slices from mice undergoing COP retrieval. In PNs, CB1 receptor antagonism did not affect COP-dependent frequency decrease. Conversely, the same treatment fully reversed mIPSCs frequency in SL cells up to the same levels of mice exposed to water alone, odor alone, sucrose alone or COA. Together with the fact that mIPSCs amplitude was not affected and that frequency values were inversely correlated with behavioral performance, these results indicate that COP retrieval is likely associated with presynaptic reduction of inhibitory transmission onto aPC SL cells. This idea is reinforced by the fact that the COP retrieval impairment under pharmacological CB1 receptor blockade is absent in mice lacking CB1 receptors from forebrain GABAergic neurons.

Hence, these results suggest that aPC-CB1 receptors control the behavioral responses induced by appetitive olfactory memory by regulating cell type-specific inhibitory transmission. More generally, they imply a dissociation between the roles of SL cells and PNs in the processing of olfactory information that will be very interesting to study in deeper details.

Interestingly, inhibitory circuits within the aPC have been shown to be strongly recruited in olfactory-dependent processes [START_REF] Poo | Odor Representations in Olfactory Cortex: "Sparse" Coding, Global Inhibition, and Oscillations[END_REF][START_REF] Franks | Recurrent Circuitry Dynamically Shapes the Activation of Piriform Cortex[END_REF][START_REF] Reuveni | Complex-learning Induced Modifications in Synaptic Inhibition: Mechanisms and Functional Significance[END_REF][START_REF] Zhan | Diverse Patterns of Odor Representation by Neurons in the Anterior Piriform Cortex of Awake Mice[END_REF]. For instance, in vivo odor exposure widely activates GABAergic interneurons in the aPC [START_REF] Poo | Odor Representations in Olfactory Cortex: "Sparse" Coding, Global Inhibition, and Oscillations[END_REF], potentially participating in the processing of odors and their meaning [START_REF] Sturgill | Somatostatin cells regulate sensory response fidelity via subtractive inhibition in olfactory cortex[END_REF]. In this context, the spatially restricted functions of CB1 receptors (i.e. on SL cells but not on PNs) suggest that CB1 receptor-dependent processes selectively tune the excitability of specific aPC principal neurons during COP retrieval. These processes would, in turn, refine the response to positively conditioned odor stimulations, eventually allowing the precise "funneling" of behavior towards preference responses.

In conclusion, this study provides a first characterization of the functional role of CB1 receptor signaling in aPC circuitry and related behaviors, thereby contributing to a better understanding of how the aPC participates in specific memory functions. S3. For supplemental information, see Figures S1 andS2. ****, p<0.0001 general solution effect. For statistical details, see Tables S1-S3. For supplemental information, see Figures S2 andS3. S1-S3. For supplemental information, see Figure S4. ns, not significant (vs Vehicle). For statistical details, see Table S1. For supplemental information, see Figure S5. S1,S3. For supplemental information, see Figures S5 andS6.

MAIN-TEXT

STAR METHODS

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY

This study did not generate new unique reagents. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Giovanni Marsicano (giovanni.marsicano@inserm.fr).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

All experimental procedures were approved by the local Committee on Animal Health and Care of Bordeaux and the French Ministry of Agriculture and Forestry (authorization number A33063098) and Committee of Ethics for Animal Welfare of the University of the Basque Country (CEEA/408/2015/Grandes Moreno, CEIAB/ 213/2015/Grandes Moreno). Two to three months-old naive male CB1-flox [mice carrying the "floxed" CB1 gene (CB1 f/f)] were used [START_REF] Marsicano | CB1 cannabinoid receptors and on-demand defense against excitotoxicity[END_REF][START_REF] Monory | The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus[END_REF][START_REF] Marsicano | The endogenous cannabinoid system controls extinction of aversive memories[END_REF]. Rescue, stop and knockout lines were generated as described [START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid CB1 receptor in cell-type-specific mutant mouse rescue models[END_REF][START_REF] Remmers | Addressing sufficiency of the CB1 receptor for endocannabinoid-mediated functions through conditional genetic rescue in forebrain GABAergic neurons[END_REF][START_REF] Ruehle | Cannabinoid CB1 Receptor in Dorsal Telencephalic Glutamatergic Neurons: Distinctive Sufficiency for Hippocampus-Dependent and Amygdala-Dependent Synaptic and Behavioral Functions[END_REF][START_REF] Marsicano | The endogenous cannabinoid system controls extinction of aversive memories[END_REF]. Briefly, Stop-CB1 mouse line was produced by silencing the endogenous CB1 gene with a loxP-flanked stop cassette in the 5′ UTR of the CB1 receptor start codon. To rescue the expression of the CB1 receptor, Stop-CB1 line was crossed with a Cre-deleter mouse line.

Conditional rescue mice were obtained by crossing Stop-CB1 mice with Dlx5/6-CRE mice (gene expressed in differentiating GABAergic neurons) allowing the expression of CB1 in GABAergic neurons, named as "GABA-CB1 rescue", and with Nex-CRE mice (gene expressed in cortical glutamatergic neurons) allowing the expression of CB1 in cortical glutamatergic neurons, named as "Glu-CB1 rescue". Total CB1 receptor knockout (CB1-KO) mice and conditional knockout animals lacking CB1 receptor in forebrain GABAergic Dlx5/6 positive neurons (GABA-CB1-KO) were obtained as described [START_REF] Monory | The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus[END_REF][START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Marsicano | The endogenous cannabinoid system controls extinction of aversive memories[END_REF]. All behavioral experiments were performed during the light phase (from 9am to 1pm) and animals were kept in individual cages under a 12h light/dark cycle (lights on 7 am) and were maintained under standard conditions with food and water ad libitum prior undergoing behavioral procedures. At least three animals from each genotype or experimental group were used for immunohistochemistry, fluorescent in situ hybridization and electrophysiology recordings.

METHOD DETAILS

Behavioral procedures

Conditioned Odor Preference (COP)

Mice were water deprived during the whole protocol. During three consecutive days, animals had 1-hour access to two bottles of water. Over the following 4 days, animals received simultaneously (1-hour access) one bottle with an odor-scented solution, either banana (isoamyl acetate, 0.05%) or almond (benzaldehyde, 0.01%) diluted in water and one bottle with a different odor-scented solution (either almond or banana) mixed with the sweet taste sucrose (0.15M, 5%). Concentrations of banana (0.05%) and almond (0.01%) solutions were chosen to be equally consumed when diluted in water and prior to any associations with other stimuli [START_REF] Busquets-Garcia | Hippocampal CB1 Receptors Control Incidental Associations[END_REF][START_REF] Busquets-Garcia | Representation-mediated Aversion as a Model to Study Psychotic-like States in Mice[END_REF][START_REF] Busquets-Garcia | Pregnenolone blocks cannabinoid-induced acute psychotic-like states in mice[END_REF]. Moreover, these almond-and banana-scented solutions were chosen to specifically served as odor cue based on previous studies indicating anosmic animals were unable to reliably detect almond-or banana-scented water (at higher concentrations than the ones used here), whereas they performed as well as control for taste detection [START_REF] Rusiniak | Flavor-illness aversions: potentiation of odor by taste in rats[END_REF][START_REF] Slotnick | Detection and discrimination of propionic acid after removal of its 2-DG identified major focus in the olfactory bulb: a psychophysical analysis[END_REF]. This provides evidence that these aqueous banana and almond compounds did not confer any behaviorally detected gustatory sensation to the drinking solution.

During COP training, the odor-scented solution present in water was named odor 1 (O1) and the other odor-scented solution associated with sucrose was named odor 2+Sucrose (O2+Sucrose). Half of the mice received banana-sucrose and the other half almond-sucrose. No differences were observed between either conditions in all the experiments performed. The position of the bottles was changed every day. After this training, a preference test was performed using a 1-hour two bottles choice: each bottle was presented with an odor-alone solution (almond versus banana diluted in water without sucrose). Subjects showing COP will drink more liquid in the bottle with the odor previously associated with sucrose (C+) than in the other bottle (C-).

In order to test the impact of CB1 receptor blockade in some experiments (Figures 1E and6J,K), mice injected with either the CB1 receptor antagonist (either AM251 in aPC or Rimonabant IP) or vehicle prior to the first COP test received 4 additional odor-sucrose pairings and were injected with the other treatment (either vehicle or CB1 receptor antagonist) before a second COP test.

Sucrose Preference

All subjects underwent 3 days habituation to water followed by 3 days with two bottles containing either water or sucrose. Finally, we evaluated the effect of aPC injection of AM251, or its vehicle, on their preference for sucrose over water.

Conditioned Odor Aversion (COA)

COA induced by gastric malaise COA using gastric malaise was adapted from previous studies [START_REF] Busquets-Garcia | Hippocampal CB1 Receptors Control Incidental Associations[END_REF][START_REF] Busquets-Garcia | Representation-mediated Aversion as a Model to Study Psychotic-like States in Mice[END_REF][START_REF] Busquets-Garcia | Pregnenolone blocks cannabinoid-induced acute psychotic-like states in mice[END_REF][START_REF] Soria-Gómez | Habenular CB1 Receptors Control the Expression of Aversive Memories[END_REF]. Mice followed the same habituation phase as described above. The conditioning phase consisted in 4 days. On days 4 and 6 the subjects received 1-hour access to odorized water (banana or almond) followed by an injection of Saline immediately after the session (O1 + sal.). On Days 5 and 7 subjects received 1-hour access to the other odor (almond or banana) that they did not receive on Days 4 and 6, followed by an injection of lithium chloride (LiCl, 0.3 M, 1% b.w.; Sigma-Aldrich; O2 + LiCl) immediately after the session. The different odors were counterbalanced between each group. After this conditioning, the subjects were given a recovery day during which they received water dispensed in two bottles during 1 hour. The following day, a preference test was performed using a 1-hour two bottles choice: each bottle was presented with an odor (almond versus banana). During the test, subjects showing COA will drink less liquid in the bottle with the odor previously associated with LiCl (C+) than in the other bottle (C-).

COA induced by quinine

The COA with quinine followed the same procedure as the COP by replacing the sucrose by 0.1mM of quinine (Sigma-Aldrich). During the test, subjects showing COA will drink less liquid in the bottle with the odor previously associated with quinine (C+) than in the other bottle (C-). An index of preference or aversion was calculated for COP or COA experiments as the following:

+ --

Surgery

Mice were anesthetized by IP injection of a mixture of ketamine (100mg/kg, Imalgene 500®) and xylazine (10mg/kg, Rompun®) or with isoflurane. Then, animals were placed into a stereotaxic apparatus (Model 900, Kopf instruments, CA, USA) with a mouse adaptor and lateral ear bars. For local deletion of CB1 receptors [START_REF] Busquets-Garcia | Hippocampal CB1 Receptors Control Incidental Associations[END_REF][START_REF] Monory | The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus[END_REF] in the aPC, CB1 flox mice were injected with an AAV-cag-CRE or its control AAV-cag-GFP (mixed serotype AAV1/AAV2, 10 10 Vg/ml) into the aPC (250µl per side, 125µl/min) with the following coordinates according to Paxinos and Franklin's mouse brain atlas [START_REF] Paxinos | The Mouse Brain in Stereotaxic Coordinates 4th Edition[END_REF]: AP +1.6, L ± 2.5, DV -4.8. For each animal receiving AAV-cag-CRE, CB1 deletion was verified by Fluorescent In Situ Hybridization against CB1 mRNA. To control that recombination did not involve the posterior PC or the anterior olfactory nucleus, data were obtained by averaging CB1 mRNA fluorescence intensity from 2/4 slices for each level in the antero-posterior axis (Figures S1H,I). In order to check region specificity across the medio-lateral axis, data were obtained from brain regions (insular cortex, aPC and lateral olfactory tubercle) bilaterally in the section for each mouse where maximal deletion of CB1 mRNA was observed (Figure 1C).

Corresponding sections were quantified in both antero-posterior and medio-lateral axes in control mice injected with AAV-cag-GFP.

For local pharmacology experiments, mice were bilaterally implanted with 3.5mm stainless steel guide cannulae (Bilaney, UK) targeting the aPC with the following coordinates [START_REF] Paxinos | The Mouse Brain in Stereotaxic Coordinates 4th Edition[END_REF]: AP +1.6, L ± 2.5, DV -4.5. Guide cannulae were secured in place with dental cement. Mice were allowed to recover for 2 weeks in individual cages before the beginning of the experiments.

The placement of aPC cannulae was determined by injection of 2% pontamine sky blue solution (0.5µl per side).

Drugs

For in vitro patch-clamp experiment, WIN 55,212-2 (5µM) (Tocris Bioscience) and AM251 (4µM) (Tocris Bioscience) were prepared in Dimethyl Sulfoxide (DMSO) and applied for 10min.

For behavioral experiments, AM251 was dissolved in a mixture of 10% Cremophor-EL, 10% DMSO and 80% saline (NaCl 0.9%). AM251 (4µg/0.5µl per side) or its vehicle was injected bilaterally in the aPC using silicone tubing connected to a peristaltic pump (PHD 22/2000 Syringe Pump Infusion, Harvard Apparatus, Massachusetts, USA, flow rate: 0.5µl/min). Rimonabant (Cayman Chemical) was dissolved in a mixture of 1.25% Tween20, 1.25% DMSO and 97.5% saline (NaCl 0.9%). Rimonabant (1 mg/kg) or its vehicle was injected intraperitoneally (IP) in a volume of 10 ml/kg.

Mice injected with AM251 (4µg/0.5µl per side) or Rimonabant (1mg/kg) were left in their home cage 10 min or 30min before bottles presentation, respectively. In order to habituate animals to receive aPC infusion and systemic injection, animals were injected with a saline solution (NaCl 0.9%) in the same manner during the two previous days. Mice receiving local aPC infusion were kept awake and maintained by the tail during the injection.

Immunohistochemistry

Mice were anesthetized with pentobarbital (Exagon, 400 mg/kg body weight), transcardially perfused with phosphate-buffered solution (PBS 0.1M, pH 7.4) before being fixed with 4% formaldehyde prepared at 4°C. Serial coronal sections were cut at 40µm and collected in PBS at room temperature (RT). Sections were permeabilized in a blocking solution of 10% donkey serum, 0.3% Triton X-100 and 0.02% sodium azide in PBS for 1 hour at RT. Free-floating sections were incubated with a goat polyclonal antibody against C-terminal sequence of the mouse CB1

receptor (1:2000, Frontier Science Co.) for 48h at 4°C. After several washes, slices were incubated for 2 hours with a secondary anti-goat antibody conjugated to Alexa 488 (1:500, Fisher Scientific) and then washed in PBS at RT. Finally, sections were incubated with DAPI (1:20 000, Fisher Scientific) for 5 minutes before being washed, mounted and coverslipped. The fluorescence was visualized with an epifluorescence Leica DM6000 microscope.

Immunocytochemistry for electron microscopy

For detailed methodological procedure see [START_REF] Puente | High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor Domains in Brain[END_REF].

Fluorescent in situ hybridization

The procedure was performed as described [START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF]. Briefly, mice were sacrificed by cervical dislocation. Their brains were extracted, frozen on dry ice and stored at -80°C until sectioning in a cryostat (14 μm, Microm HM 500M, Microm Microtech).

Fluorescein (FITC)-labeled riboprobes against mouse CB1 receptor and digoxigenin (DIG)-labeled riboprobes against mouse GAD65 were prepared as described [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF].

After hybridization overnight at 60°C with the mixture of probes, the slides were washed with different stringency wash buffers at 65°C. Then, the slides were blocked with a blocking buffer prepared according to the manufacturer's protocol. Anti-DIG or anti-FITC antibodies conjugated to horseradish peroxidase (HRP) (Roche; 1 :2000)

were applied 2 hours at RT or overnight at 4°C to detect respectively GAD65-DIG or CB1-FITC probes. Probes hybridization was revealed by a tyramide signal amplification (TSA) reaction using Cyanine 3-labeled tyramide (Perkin Elmer; 1:100 for 10 minutes) to detect GAD65 signal or FITC-conjugated tyramide (Perkin Elmer;

1:80 for 12 minutes) to amplify the signal of CB1. The slides were incubated in 4',6diamidino-2-phenylindole (DAPI; 1:20 000; FISHER Scientific) before being washed, coverslipped and visualized with an epifluorescence Leica DM6000 microscope.

Electrophysiology

All the animals were sacrificed by dislocation during the light phase (9am to 12am).

QUANTIFICATION AND STATISTICAL ANALYSIS

Behavioral data

For all the experiments, data are presented as absolute liquid intake. Considering the variability of liquid consumption during the two first days of pairings (likely due to the random choice of a bottle at day 1 and the confusion that might appear because of the inverted position of the bottles at day 2), only the last two days of the learning phase showing a reliable preference/aversion behavior were presented for each experiments.

Numerical evaluation for electron microscopy

Semi-quantitative analysis of CB1 receptor presence in excitatory or inhibitory terminals was done in aPC layers I and II of CB1-WT and CB1-KO according to our published procedure [START_REF] Puente | High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor Domains in Brain[END_REF]. Total analyzed area was more than 2200 µm 2 per genotype (n=3).

Numerical evaluation for FISH

Cells expressing mRNAs were quantified in the three layers of the aPC. Because CB1 mRNA level is variable, CB1 positive cells were classified according to the level of transcript visualized by the intensity of fluorescence [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF]. "High-CB1" cells were considered to be round-shaped and intense staining covering the entire nucleus whereas "Low-CB1" cells were defined with discontinuous shape and lowest intensity of fluorescence allowing the discrimination of grains of staining. Numerical evaluation of the double FISH was performed manually in 118 sections from 4 animals, by evaluating the coexpression of CB1-positive cells with GAD 65 marker.

Electrophysiology

Electrophysiological data were filtered at 4kHz by a Digidata 1440A (Molecular devices) and they were collected during the last 5min of recording in each condition.

Electrical properties were analyzed with Clampfit and mIPSCs were analyzed using Axograph software.

Statistics

Electrophysiological and behavioral data were analyzed with Prism Software

Figure 1 .

 1 Figure 1. CB1 receptors in the aPC are necessary for retrieval of odor

Figure 2 .

 2 Figure 2. CB1 receptors in the aPC are not involved in the retrieval of odor

Figure 3 .

 3 Figure 3. CB1 receptors are highly expressed in GABAergic interneurons in the

Figure 4 .

 4 Figure 4. Topographic distribution of CB1 receptor-positive GABAergic

Figure 5 .

 5 Figure 5. CB1 receptors control inhibitory transmission in SL cells of the aPC

Figure 6 .

 6 Figure 6. Involvement of GABAergic transmission in the CB1 receptor-

( I )

 I Correlation between the average of mIPSC frequencies in PNs from individual animals and their COP retrieval performances, expressed as Preference index (COP vehicle, n=4; COP Rim, n=5). (J-K) Consumption of the C+ and C-odor-scented solutions in (J) GABA-CB1-WT (n=31) and in (K) GABA-CB1-KO mice (n=23) receiving IP injections of either vehicle or Rim before a COP test (see STAR Methods). *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant. For statistical details, see Tables

(

  GraphPad). Repeated or unpaired statistical analyses were obtained with Student's t-test,ANOVA (one-way or two way), mixed effects analysis and linear regression to compare two or multiple groups and for correlation where appropriate. When ANOVA provided significant main factor effects or significant interactions, Dunnett or Sidak post-hoc analyses were performed as appropriate. Statistical details are presented in tables S1, S2 and S3. Significance was set at p < 0.05 and data are expressed as mean ± SEM.

  Ultrathin sections of 50 nm were collected on nickel mesh grids, stained with 2.5% lead citrate for 20 minutes, and examined in a JEOL JEM 1400 Plus electron microscope. Tissue preparations were photographed by using a digital camera coupled to the electron microscope. Adjustments in contrast and brightness were made to the figures in Adobe Photoshop (Adobe Systems, San Jose, CA).

	Coronal anterior Piriform Cortex
	(aPC) vibrosections were cut at 50 µm and collected in 0.1 M phosphate buffer (pH
	7.4) at RT. Sections were preincubated in a blocking solution of 10% BSA, 0.1%
	sodium azide, and 0.02% saponin prepared in 1X Tris-HCl-buffered saline, pH 7.4,
	for 30 minutes at RT. A pre-embedding silver-intensified immunogold method was
	used for localization of the CB1 receptor protein. Briefly, aPC sections were
	incubated with the primary goat polyclonal anti-CB1 receptor antibody (2 µg/ml
	Frontier Sciences Institute; goat polyclonal) in 10% BSA/Tris-HCl-buffered saline
	containing 0.1% sodium azide and 0.004% saponin on a shaker for 48h at 4ºC. After
	several washes in 1% BSA/Tris-HCl-buffered saline, tissue sections were incubated
	with a secondary 1.4-nm gold-labeled rabbit anti-goat Immunoglobulin G (Fab
	fragment; 1:100; Nanoprobes Inc.) in 1% BSA/Tris-HCl-buffered saline with 0.004%
	saponin on a shaker for 4 hours at RT. Sections were washed in 1% BSA/ Tris-HCl-
	buffered saline overnight at 4ºC and postfixed in 1% glutaraldehyde in Tris-HCl-
	buffered saline for 10 minutes at RT. After several washes in double-distilled water,
	gold particles were silver intensified with an HQ Silver kit (Nanoprobes Inc.) for
	approximately 12 minutes in the dark and then washed in double-distilled water first,
	and in a 0.1M phosphate buffer, pH 7.4 later. Stained sections were osmicated (1%
	osmium tetroxide, in 0.1 M phosphate buffer, pH 7.4, 20 minutes), dehydrated in
	graded alcohols to propylene oxide, and plastic-embedded in Epon resin 812.

ACKNOWLEDGMENTS

We thank all the personal of the Animal Facility of the NeuroCentre Magendie for mouse care and genotyping. We also thank Gabriel Lepousez, Antoine Nissant, Pierre-Marie Lledo and all the members of Marsicano's lab for useful discussions. This work was supported by INSERM (to G.M.), INRA (to G.F.), Fondation pour la Recherche Médicale (DRM20101220445 to G.M. and FDT20170436845 to G.T.), French State/Agence Nationale de la Recherche (LABEX BRAIN ANR-10-LABX-43 to G.M., G.F. and A.B.G., ANR-10-IDEX-03-02 to A.B.-G, NeuroNutriSens ANR-13-BSV4-0006-02 to G.M., Orups ANR-16-CE37-0010 to G.M. and G.F., CaCoVi ANR-18-CE16-0001 to G.M., MitObesity ANR-18-CE14-0029 to G.M.), EU-FP7 (PAINCAGE, HEALTH-603191 to G.M.; FP7-PEOPLE-2013-IEF-623638 to A.B.-G.), European Research Council (Endofood, ERC-2010-StG-260515, CannaPreg, ERC-2014-PoC-640923 and MiCaBra, ERC-2017-AdG-786467, to G.M.), Human Frontiers Science Program (to G.M.), Region Aquitaine (to G.M.), Fyssen Foundation (to E.S.-G.). MINECO/FEDER, UE (SAF2015-65034-R to PG), The Basque Government (BCG IT764-13 to PG), Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III) and European Regional Development Funds-European Union (ERDF-EU) (RD16/0017/0012 to P.G.).

DATA AND CODE AVAILABILITY

This study did not generate datasets/code. Further data information are available upon request by contacting the Lead Contact, Giovanni Marsicano (giovanni.marsicano@inserm.fr).

1

DECLARATION OF INTERESTS

The authors declare no competing interests.