*Review* **Cannabinoid Control of Olfactory Processes: The** *Where* **Matters**

#### **Geo**ff**rey Terral 1,2,3, Giovanni Marsicano 1,2, Pedro Grandes 4,5 and Edgar Soria-Gómez 4,5,6,\***

1 INSERM, U1215 NeuroCentre Magendie, 146 rue Léo Saignat, CEDEX, 33077 Bordeaux, France; geoffreyterral@gmail.com(G.T.);giovanni.marsicano@inserm.fr(G.M.)

 2 UniversityofBordeaux,146rue LéoSaignat,33000Bordeaux,France



Received: 25 February 2020; Accepted: 13 April 2020; Published: 16 April 2020

**Abstract:** Olfaction has a direct influence on behavior and cognitive processes. There are different neuromodulatory systems in olfactory circuits that control the sensory information flowing through the rest of the brain. The presence of the cannabinoid type-1 (CB1) receptor, (the main cannabinoid receptor in the brain), has been shown for more than 20 years in different brain olfactory areas. However, only over the last decade have we started to know the specific cellular mechanisms that link cannabinoid signaling to olfactory processing and the control of behavior. In this review, we aim to summarize and discuss our current knowledge about the presence of CB1 receptors, and the function of the endocannabinoid system in the regulation of different olfactory brain circuits and related behaviors.

**Keywords:** olfaction; endocannabinoids; olfactory epithelium; olfactory bulb; piriform cortex; CB1 receptor

#### **1. The Endocannabinoid System: A General Overview**

Cannabis sativa, also known as marijuana or cannabis, has been used for thousands of years for its therapeutic and recreational properties. Nowadays, after tobacco and alcohol, cannabis is the most commonly consumed drug of abuse, with 188 million cannabis users estimated worldwide in 2017 [1]. The cannabinoid receptors type-1 (CB1) and type-2 (CB2), their endogenous ligands (endocannabinoids), and the synthetic and degradative enzymes that regulate endocannabinoid levels support the concept of the endocannabinoid system (ECS) as participating in the regulation of physiological processes [2]. CB1 and CB2 receptors belong to the superfamily of G-protein-coupled receptors (GPCRs) that consist of seven transmembrane domains with an extracellular N-terminal and an intracellular C-terminal tail [3]. At the synaptic level, endocannabinoids can be synthesized, but not exclusively [4], by post-synaptic intracellular calcium elevations, which can be caused by various stimuli, including depolarization, the activation of metabotropic acetylcholine, and glutamate receptors, particularly Gq-coupled receptors (i.e., M1/M3 and mGluR 1/5) [3]. Once produced, endocannabinoids act on CB1 receptors that are mainly described at pre-synaptic terminals [2,3,5], and other cellular locations [6].

In neurons, the main effect of CB1 receptor activation is a decrease in neurotransmitter release, inducing different forms of endocannabinoid-mediated plasticity [3], such as the depolarizationinduced suppression of inhibition/excitation (DSI/DSE; [7–9]), or the long-term depression of inhibitory/

excitatory synapses [10–14]. CB1 receptors are widely expressed in the central nervous system and likely represent the most abundant GPCR in the brain [15]. Given its ubiquitous expression in multiple brain areas, CB1 receptors modulate a variety of functions, from sensory perception to more complex cognitive processes such as learning and memory [16–18].

#### **2. Role of the Endocannabinoid System in Olfactory Circuits**

Known for a long time, one of the predominant subjective effects of cannabis intoxication is the alteration of sensory perception, including olfactory processes [19]. However, although relatively high levels of CB1 receptors were described in the 1990s in many olfactory brain areas of rodents [20–23], their olfactory-related functions only started to be studied during the last decade. Notably, the involvement of CB1 receptors in specific odor-related processes has been reported in specialized olfactory structures such as the olfactory epithelium (OE; [24–27]), the main olfactory bulb (MOB; [18,28–34]), and the piriform cortex (PC; [35–40]), but also in other brain areas processing olfactory information [41–44]. For the sake of clarity in this review, we will focus on describing the role of the ECS, particularly CB1 receptor signaling, in specific main olfactory areas (i.e., OE, MOB, and PC).

#### **3. The Endocannabinoid System in the Olfactory Epithelium**

The first hypothesis for the physiological involvement of endocannabinoids in olfactory processes came from three observations: (1) The olfactory perception was shown to be changed depending on the feeding state of individuals [45,46], (2) the ECS were proposed to be involved in food intake [18,47], and (3) the anatomical and functional connectivity between peripheral organs regulating energy balance and olfactory structures [18,48]. Czesnik, Breunig, and colleagues [24,25] provided the first evidence that cannabinoids could modulate olfaction. These studies revealed the presence of CB1 receptors in the olfactory sensory neurons (OSN) of Xenopus laevis and demonstrated that endocannabinoids modulate odor-evoked responses. Additionally, they found that the production of endocannabinoids depends on the hunger state of the animal, which is responsible for changes in odor sensitivity activity. Similarly, CB1 receptors were also found in the OSN of rodents [27]. The CB1 receptor agonists changed odorant-induced cellular activity, but the authors did not observe olfactory consequences in the behavior of mutant mice lacking CB1 receptors (CB1-KO; [27]). Despite the species differences, several divergences appear between these studies. For instance, the first two studies evaluated the impact of cannabinoids on odor sensitivity by recording the cellular activity of the OSN with calcium imaging and electrophysiological methods [24,25]. Instead, Hutch and colleagues [27] investigated the involvement of CB1 receptors in olfactory-mediated learning and memory tasks such as the buried food test and a habituation/dishabituation paradigm. In addition, CB1-KO mice lack brain specificity and might be confounded by compensatory mechanisms [49]. Thus, the physiological role of CB1 receptors in the mammalian OE still remains unclear and will need further investigation.

#### **4. The Endocannabinoid System in the Olfactory Bulb**

In the mammalian MOB, the ECS was first described as a modulator of GABAergic transmission [28,33]. Pharmacological approaches, combined with in vitro patch-clamp experiments, highlighted that CB1 receptors modulate the firing pattern of periglomerular (PG) and external tufted cells (eTCs). Considering that PG cells form synapses with mitral and tufted cells [50], CB1 signaling may indirectly regulate the main output activity of the MOB neurons. Indeed, the inhibitory inputs of eTCs display spontaneous DSI [33], and the pharmacological manipulation of CB1 receptor signaling modulates mitral cell activity, likely through indirect control of inhibitory transmission [34]. These results sugges<sup>t</sup> that endocannabinoids are capable of controlling mitral/tufted cell activity through the CB1 receptors on PG cells. Although the authors did not investigate the behavioral impact of these effects, the CB1 receptors' activation may increase the signal-to-noise ratio and, thus, the overall sensitivity of the glomerulus to sensory inputs. Moreover, CB1 receptors are present in glutamatergic corticofugal fibers (CFF) coming from projection neurons from anterior cortical olfactory areas (including the

anterior olfactory nucleus, AON, and the anterior piriform cortex), and targeting granule cells (GCs) of the MOB [32]. Consistent with the idea that cannabinoid signaling in the olfactory system might control the feeding state of the organism, the hypophagic phenotype observed in mice lacking CB1 receptors in their glutamatergic neurons is associated with an increased activity of CFF onto GCs. Notably, endocannabinoid levels increase in the MOB during fasting, allowing for the dampening of the excitation of GCs. Given that GCs control mitral cell activity, CB1 receptor activation of CFF induces the disinhibition of mitral cells. This e ffect is followed by a fasting-related enhancement in olfactory sensitivity, which correlates with the amount of food ingested upon refeeding. These results sugges<sup>t</sup> that the endocannabinoid-mediated regulation of olfactory output information controls olfactory perception and food intake [32]. Since CB1 receptors have been described as being expressed on CFF fibers, they may thus regulate all of the downstream synapses of these fibers. This hypothesis was recently verified in the synapse between the CFF and the so-called deep short axon cells (dSAs; [31]). Indeed, depolarization of dSAs in the MOB of mice elicits pre-synaptic CB1 receptors' transient suppression of excitatory CFF inputs (DSE). In addition, the authors demonstrated that dSAs could inhibit GCs, thereby suppressing GC to mitral cells inhibition. Interestingly, depending on the CFF synaptic strength, the CB1 receptor signaling can either control the synapses from dSAs to GCs, or directly from GCs to mitral cells, suggesting a double dissociation in the control of olfactory bulb output neurons [31]. However, the behavioral consequences of this bidirectional e ffect remain to be elucidated.

#### **5. The Endocannabinoid System in the Piriform Cortex**

The PC is a brain area capable of generating epileptiform activity [51]. In other brain structures such as the hippocampus, CB1 receptors have been shown to protect against seizures [52,53]. Thus, the anticonvulsant e ffects of cannabinoids were assessed in PC slices [36]. The authors demonstrated that CB1 receptor agonists reduce seizures, indicating that CB1 receptor activation is able to control PC activity [36]. However, there is currently no functional evidence about how the ECS could a ffect olfactory processes under pathological conditions such as epilepsy. Furthermore, the ECS in the PC indirectly a ffects social behavior [38]. Although it does not a ffect social interactions per se, local injections of a CB1 receptor antagonist into the posterior PC (pPC) reversed the impairment of social sni ffing time induced by an activation of dopamine receptors, suggesting that the ECS in the pPC has a deleterious e ffect on social behavior when coupled with dopamine activation [38]. Moreover, the PC is an important area involved in olfactory memory [54]. Considering that the ECS is highly studied in learning and memory functions [16], other studies investigated its role in PC-dependent olfactory learning and memory. In the pPC, odor-discrimination training leads to the endocannabinoid-mediated modification of inhibitory synapses [35]. Indeed, the learning of a complex olfactory rule induces the activation of CB1 receptors, which in turn enhances GABAergic conductance in post-synaptic pPC pyramidal neurons, indicating a postsynaptic e ffect [35]. Despite the possible post-synaptic CB1 receptors' localization, or that endocannabinoids can modulate directly postsynaptic GABAergic receptors [55], further experiments will determine how CB1 receptor activation allows for controlling GABAergic conductance in the pPC. In the anterior PC (aPC), CB1 receptors were mainly described at GABAergic synapses, where they modulate inhibitory transmission and plasticity [37,40]. Moreover, depending on CB1 receptors in the aPC, the retrieval of appetitive, but not aversive, olfactory memory is associated with a modulation of local inhibitory transmission onto specific principal cells in the aPC [37]. These data indicate that CB1 receptors in the aPC selectively control olfactory memory retrieval related to positively motivated behaviors. Thus, it will be crucial to determine if cannabinoid signaling controls functional connection between the PC and brain regions controlling a ffective states such as the orbitofrontal cortex, the nucleus accumbens or amygdala. In fact, there is compelling literature demonstrating the participation of these brain areas in olfactory processes [56–58]. In line with this idea, recent observations in humans highlight that the state-dependent enhancement of endocannabinoid levels changes dietary choices toward high-energy food items. Interestingly, this phenomenon was related to an increase in odor responses in the PC [39].
