Keywords

1 Introduction to Cannabinergic Cerebellar Circuitry

It is well known that eCBs, principally 2-AG (Szabo et al. 2006), are released “on-demand” from Purkinje cells (PCs), the neuronal element which represents the sole controlling output of the cerebellar cortex. 2-AG is released predominantly from PC dendrites to act retrogradely on CB1Rs expressed on presynaptic axons, including excitatory parallel fibers (PF), climbing fibers, and inhibitory basket cell interneurons (INs), and stellate cells (Kawamura et al. 2006; Rodríguez-Cueto et al. 2014a; Stephens 2016a; Fig. 34.1). CB1Rs are the most prominently expressed G protein-coupled receptors in the cerebellum, and indeed in the CNS (Herkenham et al. 1991). These features combine to afford the cannabinergic circuitry a unique and privileged role in controlling cerebellar function. CB1Rs are known to play a major role in long-term plasticity at PF-PC synapses (Carey et al. 2011), proposed to be critical for cerebellar learning (Ito 1972). The role of presynaptic CB1R signaling in short-term plasticity in the cerebellum is well studied using protocols such as depolarization-induced suppression of inhibition (DSI) or depolarization-induced suppression of excitation (DSE) (Kreitzer and Regehr 2001). It has been shown that presynaptic CB1R expression can be regulated by physiological synaptic activity patterns and that such activity is linked to the regulation of eCB levels by degradative enzymes (Yang et al. 2019); this plasticity is proposed to be important for associative learning paradigms. Genetic deletion of CB1R causes impairment of fine motor control, rather than gross changes in motor function, and also impairs cerebellar development (Kishimoto and Kano 2006; Martinez et al. 2020). Endogenous or exogenous activation of CB1Rs, the latter for example by Δ9-THC in cannabis, developmentally regulates synaptic strength and network activity (Barnes et al. 2020). In particular, CB1R expression has been reported to undergo a development switch, with pronounced but transient expression at presumptive mossy fiber afferent terminals in the cerebellum of newborn rodents, prior to the establishment of the well-described dominant CB1R expression on excitatory or inhibitory afferents to PCs in the adult cerebellum (Barnes et al. 2020). Glial cell elements, including astrocytes and microglial, are also reported to express CB1R within the cerebellar cortex (Rodríguez-Cueto et al. 2014a). Such studies point to the critical importance not only of cannabinergic pathways in the cerebellum but also to the potential of illicit cannabis use to disrupt cerebellar circuitry and cause deficits in function. At a functional level, we have shown that du2J “ducky” ataxic mice have deficits in CB1R-mediated signaling that could contribute to disease phenotype (Wang et al. 2013). Disruptions in eCBS signaling have been implicated in cerebellar disease states including SCA2 and SCA3 (Kasumu and Bezprozvanny 2012; Rodríguez-Cueto et al. 2016). In a similar manner, a significant reduction in CB1R protein expression within the cerebellum has also been reported in a mouse model of Dravet syndrome (DS) carrying a knock-in missense mutation in the Scn1a gene (which is defective in DS) (Satta et al. 2021); these deficits are correlated with altered, potentially cerebellar-related, behaviors. At a human level, the partial CB1R agonist Δ9-THC is well known to mediate the “high” associated with illegal cannabis use. While there are some proponents that advocate the benefits of medicinal cannabis use, it is much less clear if Δ9-THC is the cannabis component that mediates any proposed benefit(s). The general medical consensus is, rather, that Δ9-THC is deleterious for CNS and cerebellar function. For example, neuroimaging studies examining effects of exogenous cannabis reported reductions in cerebellar volume, activity patterns, and deficits in cerebellar-dependent working memory and learning (Blithikioti et al. 2019); moreover, these deficits were correlated positively with heavy cannabis use in adolescents.

Fig. 34.1
A schematic diagram of the cerebellar endocannabinoid system displays the components of the negative allosteric antagonist, cannabinoid receptor 1 antagonist, astrocyte, parallel fiber, interneuron, dendritic spine, F A A H inhibitor, and glutamate.

Cerebellar endocannabinoid system and potential pharmacological targets. In Purkinje cell postsynaptic dendritic spines, 2-AG is synthesized by DAGLα from DAG. 2-AG is released retrogradely to act on presynaptic CB1Rs at excitatory PF and inhibitory IN terminals to suppress release of glutamate (Glu) or GABA, respectively. Astrocytes and microglial also express CB1Rs. PCs also express CB2Rs, predominantly in soma. 2-AG is degraded by MGL, produced in presynaptic PF terminals, and also in astrocytes. FAAH is expressed in PC soma. Red boxes indicate potential pharmacological intervention strategies

Full size image

CB2Rs may also contribute to eCBS effects in the cerebellum. In general, the roles of CB2Rs in other CNS regions have come under recent research focus. Within the cerebellum, studies have reported some contradictory findings regarding CB2R expression; however, functional electrophysiological data support the role of CB2R in cerebellar circuitry. Thus, Sadanandan et al. (2020) reported a variable expression of CB2R in PC soma from juvenile mice and, moreover, that exogenous CB2R agonists can reduce evoked inhibitory (but not excitatory) transmission at PCs. Of further interest was that CB2R-mediated responses were postsynaptic in origin and that DSI was entirely dependent on CB1Rs, and not CB2Rs. Hence, it appears that CB2R signaling differs from that of CB1R, in that it is not mediated by retrograde signaling by PC-derived eCBs. The authors suggest that CB2R signaling may be more relevant under conditions of sustained eCB release or, potentially, when activated by exogenous agents such as Δ9-THC (Sadanandan et al. 2020). Some further support for a potential pathophysiological role of CB2Rs in the cerebellum is the report of elevated CB2R expression postmortem in patients with SCAs (Rodríguez-Cueto et al. 2014a); of further interest, this increase in CB2R expression was reported to be co-incident with that of CB1R, suggesting a potential symbiotic change in eCBS signaling during cerebellar disease.

2 Metabolic Control of the eCBS

eCBS function is intimately controlled by a series of metabolic enzymes. 2-AG is synthesized from diacylglycerol (DAG) by the lipase DAGLα in PC postsynaptic dendritic spines and is degraded by serine hydrolases, predominantly monoacylglycerol lipase (MGL), produced in presynaptic terminals and also in astrocytes (Yoshida and Fukaya 2006; Tanimura et al. 2012; Viader et al. 2015; Stephens 2016a; Fig. 34.1). Work using conditional MGL knockout mice has demonstrated that neuronal and astrocytic cells act co-operatively to regulate eCB-mediated retrograde synaptic depression in the cerebellum (Viader et al. 2015). Moreover, Chen et al. (2016) have shown that neurons and astrocytes combine effectively to regulate spatial 2-AG levels, limiting distribution and, hence, synapse-specific signaling within the cerebellum. While 2-AG is recognized as the most prominent eCB in the cerebellum, there is evidence that the enzyme fatty acid amide hydrolase (FAAH), which acts to degrade the eCB anandamide, is expressed throughout the cerebellum, in particular in PCs, cerebellar nuclei and the molecular layer (Suárez et al. 2008). Thus, there is potential to target a series of enzymes, including DAGLα, MGL, and FAAH to modulate eCB tone (see Fig. 34.1). In this regard, levels of degrative MGL and FAAH have both been reported to be increased in postmortem cerebellar tissue in patients with SCAs (Rodríguez-Cueto et al. 2014b).

3 Therapeutic Targeting of the eCBS in the Cerebellum

The cerebellum frequently overcomes its “little brain” status and is now recognized for its importance in cognitive and emotional learning and neurodevelopment. By extension, deficits in cerebellar circuitry can lead to a range of psychopathologies and cognitive disorders, including SCAs, autism, schizophrenia, and attention deficit and hyperactivity disorders (Stephens 2016a; Stoodley 2016; Hariri 2019). Knowledge of the different elements within the eCBS may be exploited to develop therapeutic agents. General pharmacological strategies are also summarized in Fig. 34.1. These strategies include the use of CB1R antagonist/inverse agonists such as prototypic rimonabant; such agents most likely work by reducing constitutive endocannabinergic tone via an inverse agonism action. However, rimonabant, introduced as an anti-obesity agent, was subsequently withdrawn amid post-marketing identification of potential adverse psychiatric effects. The therapeutic targeting of different eCB enzymes has also been explored. For example, the DAGLα inhibitor, orlistat, is an anti-obesity agent that targets the gastrointestinal tract. However, the FAAH inhibitor, BIA 10–247, under investigations for various central indications including anxiety and Parkinson’s disease as well as for anti-obesity potential, was another high-profile case where serious adverse events, including the death of one volunteer, resulted in termination of human trials. Overall, reports of adverse central effects have somewhat curtailed drug discovery in this area, although peripherally acting drugs are still under investigation and such avenues may lead to improved safety profiles and re-ignite this area.

A compound of on-going therapeutic interest is CBD. CBD is licensed to treat severe childhood epilepsies (Williams and Stephens 2020) and has potential to treat cerebellar diseases including SCAs (Stephens 2016b). Although an exact mechanism of action is still under debate, CBD has potential to modulate the eCBS via different proposed mechanisms, including a negative allosteric antagonism of CB1Rs and antagonism of CB2Rs. In general, there is now good evidence that CBD, rather than Δ9-THC, mediates many of the proposed beneficial effects of medicinal cannabis. Indeed, CBD is reported to ameliorate the effects of the CB1R partial agonist Δ9-THC, including in cerebellar tissue (Whalley et al. 2019), and may act to limit effects of endogenous cannabinoid agonists in a similar manner (e.g., Hohmann et al. 2019). Functional magnetic resonance imaging studies have shown that CBD decreases blood oxygen level-dependent signaling in the mammalian cerebellum (Sadaka et al. 2021), consistent with a general inhibition of activity; such a mechanism may support CBD positive therapeutic effects on disease states linked to over activity of the cerebellar circuitry.

Overall, diseases of the cerebellum continue to be ripe for therapeutic invention involving the eCBS and there are clear opportunities to exploit the critical contribution of the eCBS to cerebellar circuitry, in particular output of PCs, using different pharmacological strategies. However, lessons will need to be learned from the identification of different adverse effects associated with some high-profile therapeutic failures in order to progress this area over the next few years.