Endocannabinoid Signaling and Synaptic Function, Pablo E. Castillo et al., 2012

Endocannabinoid Signaling and Synaptic Function

Pablo E. Castillo, Thomas J. Younts, Andres E. Chavez, and Yuki Hashimotodani

Neuron, Cell Press, 2012.



Endocannabinoids are key modulators of synaptic function. By activating cannabinoid receptors expressed in the central nervous system, these lipid messengers can regulate several neural functions and behaviors. As experimental tools advance, the repertoire of known endocannabinoid-mediated effects at the synapse, and their underlying mechanism, continues to expand. Retrograde signaling is the principal mode by which endocannabinoids mediate short- and long-term forms of plasticity at both excitatory and inhibitory synapses. However, growing evidence suggests that endocannabinoids can also signal in a nonretrograde manner. In addition to mediating synaptic plasticity, the endocannabinoid system is itself subject to plastic changes. Multiple points of interaction with other neuromodulatory and signaling systems have now been identified. In this Review, we focus on new advances in synaptic endocannabinoid signaling in the mammalian brain. The emerging picture not only reinforces endocannabinoids as potent regulators of synaptic function but also reveals that endocannabinoid signaling is mechanistically more complex and diverse than originally thought.



Since the discovery of D9-tetrahydrocannabinol (THC) as the main psychoactive ingredient in marijuana, and the cloning of cannabinoid receptors and the identification of their endogenous
ligands (endocannabinoids [eCBs]), our understanding of the molecular basis and functions of the eCB signaling system has evolved considerably. Extensive research in the last 15 years has consolidated our view on eCBs as powerful regulators of synaptic function throughout the CNS. Their role as retrograde
messengers suppressing transmitter release in a transient or long-lasting manner, at both excitatory and inhibitory synapses, is now well established (Alger, 2012; Chevaleyre et al., 2006; Freund et al., 2003; Kano et al., 2009; Katona and Freund, 2012). Apart from signaling in more mature systems, the eCB
system has been implicated in synapse formation and neurogenesis (Harkany et al., 2008). It is also widely believed that by modulating synaptic strength, eCBs can regulate a wide range of neural functions, including cognition, motor control, feeding behaviors, and pain. Moreover, dysregulation of the eCB system is implicated in neuropsychiatric conditions such as depression and anxiety (Hillard et al., 2012; Mechoulam and Parker, 2012). As such, the eCB system provides an excellent opportunity for therapeutic interventions (Ligresti et al., 2009; Piomelli, 2005). Their prevalence throughout the brain suggests that eCBs are fundamental modulators of synaptic function. This Review focuses on recent advances in eCB signaling at central synapses.

The eCB signaling system comprises (1) at least two Gprotein coupled receptors (GPCRs), known as the cannabinoid type 1 and type 2 receptors (CB1R and CB2R); (2) the endogenous ligands (eCBs), of which anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the best characterized; and (3) synthetic and degradative enzymes and transporters that regulate eCB levels and action at receptors. An enormous amount of information on the general properties of the eCB system has accumulated over the last two decades (for general reviews on the eCB system, see Ahn et al., 2008; Di Marzo, 2009; Howlett et al., 2002; Pertwee et al., 2010; Piomelli, 2003). We discuss essential features of this system in the context of synaptic function.

The principal mechanism by which eCBs regulate synaptic function is through retrograde signaling (for a thorough review, see Kano et al., 2009). Here, postsynaptic activity leads to the production of an eCB that moves backward across the synapse, binds presynaptic CB1Rs, and suppresses neurotransmitter
release (Figure 1A). However, there is also evidence suggesting that eCBs signal in a nonretrograde or autocrine manner, in which they can modulate neural function and synaptic transmission by engaging transient receptor potential vanilloid receptor type 1 (TRPV1) and also CB1Rs located on or within the postsynaptic cell (Figure 1B). Finally, recent studies indicate that eCBs can signal via astrocytes to indirectly modulate presynaptic or postsynaptic function (Figure 1C). This Review aims to highlight the emerging mechanistic diversity of synaptic eCB signaling.

Retrograde Endocannabinoid Signaling

The first demonstration of retrograde eCB signaling came from the discovery that eCBs mediate forms of short-term synaptic plasticity known as depolarization-induced suppression of inhibition (DSI) (Ohno-Shosaku et al., 2001; Wilson and Nicoll, 2001) and depolarization-induced suppression of excitation (DSE) (Kreitzer and Regehr, 2001). Shortly after it was shown that eCBs also mediate presynaptic forms of long-term depression (eCB-LTD) at both excitatory (Gerdeman et al., 2002; Robbe et al., 2002) and inhibitory (Chevaleyre and Castillo, 2003; Marsicano et al., 2002) synapses. eCBs have since emerged as the best characterized retrograde messengers (Regehr et al., 2009), with numerous examples of short- and long-term forms of synaptic plasticity reported throughout the brain (Heifets and Castillo, 2009; Kano et al., 2009).

CB1/CB2 receptors are Gi/o protein-coupled receptors that mediate almost all effects of exogenous and endogenous cannabinoids. CB1Rs are one of the most widely expressed GPCRs in the brain (Herkenham et al., 1990). Their localization to neuronal terminals (Katona et al., 1999, 2006) strongly suggests that CB1Rs play important roles in regulating synaptic function. Indeed, CB1R activation inhibits neurotransmitter release at synapses through two main mechanisms (Figure 2) (Chevaleyre et al., 2006; Freund et al., 2003; Kano et al., 2009). For short-term plasticity, in which CB1Rs are activated for
a few seconds, the mechanism involves direct G protein-dependent (likely via the bg subunits) inhibition of presynaptic Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) (Brown et al., 2003; Kreitzer and Regehr, 2001; Wilson et al., 2001). For long-term plasticity, the predominant mechanism requires
inhibition of adenylyl cyclase and downregulation of the cAMP/ PKA pathway via the ai/o limb (Chevaleyre et al., 2006; Heifets and Castillo, 2009). Moreover, CB1Rs only need to be engaged during the induction, but not expression, phase of eCB-LTD. Induction also requires combined presynaptic firing with CB1R activation, thereby providing a mechanism for input specificity; that is, only active synapses detecting eCBs express long-term plasticity (Heifets et al., 2008; Singla et al., 2007). The expression mechanism for eCB-LTD may involve presynaptic proteins Rab3B/RIM1a (Chevaleyre et al., 2007; Tsetsenis et al., 2011) or a reduction of P/Q-type VGCCs (Mato et al., 2008). While other effectors downstream of CB1Rs have been described, mainly in cultured cells and expression systems (Howlett, 2005; Pertwee et al., 2010), their role in regulating synaptic function is presently less clear. In contrast to CB1Rs, which are widely expressed in the brain, CB2Rs are typically found in the immune system and
are poorly expressed in the CNS. Although recent studies support a role for these receptors in the CNS (den Boon et al., 2012; Van Sickle et al., 2005; Xi et al., 2011), when compared with CB1Rs, much less is known about the precise cellular mechanism(s) and contributions of CB2Rs to brain function.

Although several eCBs have been identified, just two, AEA and 2-AG, emerged as the most relevant and prevalent regulators of synaptic function. While 2-AG seems to be the principal eCB required for activity-dependent retrograde signaling, the relative contribution of 2-AG and AEA to synaptic transmission is still debated. Functional crosstalk between 2-AG and AEA signaling was reported (Maccarrone et al., 2008), and recent findings suggest that 2-AG and AEA can be recruited differentially from the same postsynaptic neuron, depending on the type of presynaptic activity (Lerner and Kreitzer, 2012; Puente et al., 2011). A more complete signaling profile for 2-AG and AEA—including production, target identification, and degradation—is indispensable for better understanding their short- and long-term impact on synaptic function.

Synthesis and degradation of eCBs help shape their spatiotemporal signaling profile. For retrograde eCB signaling, postsynaptic neuronal depolarization elevates intracellular Ca2+ via VGCCs and elicits 2-AG production presumably by activating Ca2+-sensitive enzymes. In addition, glutamate release onto postsynaptic group I metabotropic glutamate receptors (I mGluRs) (Maejima et al., 2001; Varma et al., 2001) can generate 2-AG by activating the enzyme phospholipase Cb (PLCb) (for a review, see Hashimotodani et al., 2007a). Most likely, Ca2+ influx through VGCCs and downstream signaling from I mGluR activation converge on the same metabolic pathway to mobilize 2-AG (Figure 2A). PLCb is thought to act as a coincidence detector for postsynaptic Ca2+ and GPCR signaling (Hashimotodani et al., 2005; Maejima et al., 2005). This interaction might be important for integrating synaptic activity (Brenowitz and Regehr, 2005). On the other hand, it is worth noting that activation of I mGluRs is sufficient to mobilize eCBs to trigger short- and long-term forms of plasticity (Chevaleyre et al., 2006). For long-term plasticity, a few minutes of CB1R stimulation is needed, which can result from a brief postsynaptic I mGluR activation triggering a relatively longer-lasting 2-AG mobilization (Chevaleyre and
Castillo, 2003). Of general physiological relevance, many other Gq/11-GPCRs are known to promote eCB synthesis (Katona and Freund, 2012). Upon activation, PLCb hydrolyzes phosphatidylinositol to generate diacylglycerol, which is converted to 2-AG by diacylglycerol lipase a (DGLa). DGLa is specifically localized to postsynaptic compartments (Katona et al., 2006; Lafourcade et al., 2007; Nomura et al., 2007; Yoshida et al., 2006). Whereas pharmacological studies inconsistently implicated DGLa in short-term synaptic plasticity, genetic deletion of DGLa indicates that this enzyme is required for Ca2+-dependent 2-AG production and short- and long-term eCB-dependent synaptic plasticity (Gao et al., 2010; Tanimura et al., 2010; Yoshino et al., 2011). Once synthesized, 2-AG travels backward across the synapse; however, the precise mechanism by which this occurs is still unresolved.