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Type 1 cannabinoid receptor (CB1) is expressed in different neuronal populations in the mammalian brain. In particular, CB1 on GABAergic or glutamatergic neurons exerts different functions and display different pharmacological properties in vivo. This suggests the existence of neuron-type specific signalling pathways activated by different subpopulations of CB1. In this study, we analysed CB1 expression, binding and signalling in the hippocampus of conditional mutant mice, bearing CB1 deletion in GABAergic (GABA-CB1-KO mice) or cortical glutamatergic neurons (Glu-CB1-KO mice). Compared to their wild-type littermates, Glu-CB1-KO displayed a small decrease of CB1 mRNA amount, immunoreactivity and [³H]CP55,940 binding. Conversely, GABA-CB1-KO mice showed a drastic reduction of these parameters, confirming that CB1 is present at much higher density on hippocampal GABAergic interneurons than glutamatergic neurons. Surprisingly, however, saturation analysis of HU210-stimulated [35S]GTPγS binding demonstrated that ‘glutamatergic’ CB1 is more efficiently coupled to G protein signalling than ‘GABAergic’ CB1. Thus, the minority of CB1 on glutamatergic neurons is paradoxically several fold more strongly coupled to G protein signalling than ‘GABAergic’ CB1. This selective signalling mechanism raises the possibility of designing novel cannabinoid ligands that differentially activate only a subset of physiological effects of CB1 stimulation, thereby optimizing therapeutic action.
Type 1 cannabinoid receptor (CB1) is one of the most abundant G protein-coupled receptors in the mammalian brain with highest expression levels in the cerebellum, basal ganglia, cortex and limbic system (Marsicano and Kuner 2008). CB1 generally couples to Gi/o proteins to inhibit adenylyl cyclase (Howlett and Fleming 1984). Further intracellular effects include regulation of numerous kinases, transcription factors and ion channels (Demuth and Molleman 2006; Bosier et al. 2010). These intracellular effects collectively drive CB1′s cellular functions, of which the most prominent is the retrograde inhibition of transmitter release (Kano et al. 2009). Several studies demonstrated that CB1 activation inhibits the release of glutamate, GABA, glycine, acetylcholine, norepinephrine, dopamine, serotonin and cholecystokinin (Kano et al. 2009).
In the cortical regions of the mammalian brain, CB1 is mainly expressed in two major neuronal populations, GABAergic interneurons and glutamatergic principal neurons. There is a robust difference between the expression levels of CB1 in these two types of neurons. GABAergic interneurons express very high levels of CB1, whereas glutamatergic neurons express low to moderate levels (Marsicano and Lutz 1999). This implies that the most important inhibitory and stimulatory neurotransmitters in the cortex are, at least partially, under the control of the same regulatory system.
To dissect the physiological roles of these various receptor populations, we have generated several conditional mutant mice in which CB1 is specifically deleted in a particular neuronal population (Marsicano et al. 2003; Monory et al. 2006, 2007). Two of these mouse lines, Glu-CB1-KO and GABA-CB1-KO (Monory et al. 2006), are particularly interesting to study the differential impact of CB1-dependent control of cortical glutamatergic and GABAergic neurons.
Indeed, studies with these mice provided ample evidence about the involvement of these two populations of CB1 in mammalian brain physiology. Interestingly, contrary to general expectations, CB1 on glutamatergic cells, though much lower in numbers, produced in some cases more pronounced effects than their counterparts on GABAergic cells. Thus, behavioural analysis of Glu-CB1-KO and GABA-CB1-KO showed strong involvement of CB1 on glutamatergic cells in functions partly or fully dependent on hippocampus, such as protection against excitotoxic insults (Monory et al. 2006), stress response (Steiner et al. 2008), fear coping, stress and anxiety (Jacob et al. 2009; Kamprath et al. 2009; Dubreucq et al. 2012; Metna-Laurent et al. 2012). Similarly, CB1 on glutamatergic cells plays an important role in feeding behaviour (Lafenêtre et al. 2009; Bellocchio et al. 2010) and brain development (Mulder et al. 2008). Conversely, CB1 expressed on GABAergic neurons seems to be dispensable for several functions of endocannabinoid signalling or bears a completely opposite impact on these functions (Monory et al. 2006; Lafenêtre et al. 2009; Bellocchio et al. 2010; Dubreucq et al. 2012; Metna-Laurent et al. 2012). These observations might be simply because of the inhibition by CB1 activation of both excitatory and inhibitory neurotransmission, which logically will lead to different network effects. However, the administration of exogenous CB1 agonist to conditional mutant mice suggested that the CB1 pools expressed on the two neuronal populations likely possess different pharmacological properties. Thus, GABAergic neurons are not involved in the so-called tetrad effect induced by cannabinoids, which is mainly mediated by ‘glutamatergic’ CB1 (Monory et al. 2007). Interestingly, cannabinoids often exert biphasic effects on several behaviours, with low and high doses resulting in opposite effects (Sulcova et al. 1998; Chaperon and Thiébot 1999; Tzavara et al. 2003). We recently found that CB1 on glutamatergic cells is responsible for the effects of low doses of agonists, while CB1 on GABAergic cells responds only to higher doses (Bellocchio et al. 2010; Rey et al. 2012).
Altogether, these data suggest that CB1 on GABAergic or glutamatergic neurons might differ in its binding and/or signalling properties. Using hippocampal protein extracts from conditional mutant mice, we describe here neuronal type-specific G protein signalling that might provide mechanistic explanation for differential functions of CB1 and for the biphasic effects of cannabinoids.
- Top of page
- Materials and methods
CB1 is expressed in different neuronal populations in the mammalian brain (Marsicano and Lutz 1999; Tsou et al. 1999; Monory et al. 2006). To dissect the physiological roles of these various receptor populations, we have generated several conditional mutant mice in which CB1 is deleted in specific neuronal populations (Marsicano et al. 2003; Monory et al. 2006, 2007). As most glutamatergic principal neurons and many GABAergic interneurons express CB1 in cortical areas, two of these lines, Glu-CB1-KO and GABA-CB1-KO, were chosen for this project to study hippocampal CB1 expression, binding and G protein signalling in GABAergic versus glutamatergic cells.
In the hippocampal formation, mainly two distinct cell populations express CB1 mRNA: CCK+ GABAergic interneurons express high levels of CB1 mRNA (Marsicano and Lutz 1999), while glutamatergic principal cells (pyramidal neurons and mossy cells but not granular cells) express low amounts (Marsicano and Lutz 1999; Marsicano et al. 2003; Monory et al. 2006). CB1 is an axonal protein and for this reason there is a specific difference between mRNA and protein distribution, as the mRNA is located in the perinuclear soma. Consistently to this notion, intense CB1 protein staining is visible at sites where basket cell terminals surround pyramidal and granular cells. Somewhat weaker signal is visible in the strata molecularis > radiatum and > oriens, where the CB1 signal is predominantly originating from the interconnecting network of inhibitory interneurons innervating the dendritic arbour of pyramidal or granular cell bodies (Katona et al. 1999, 2001; Egertová and Elphick 2000). CB1 staining of glutamatergic terminals amounts to a much weaker and diffuse signal most prominently visible at the inner third of the stratum molecularis (Katona et al. 2006; Kawamura et al. 2006; Monory et al. 2006). Apart from these two major components of the CB1 population of the hippocampus, however, extrahippocampal neurons contribute to this population, too. Several regions send projecting neurons to the hippocampus, some of them are CB1+. Most important of these are the inputs from the entorhinal cortex which are glutamatergic pyramidal cells and are terminating in all parts of the hippocampus (Shepherd, 1990). Glutamatergic inputs arrive from subcortical regions too – hypothalamic projections terminate mostly in the stratum molecularis and CA3. In addition, neurons of several other neurotransmitter systems send their axons to the hippocampus. Notably, cholinergic neurons from the septum (Nyíri et al. 2005), serotonergic neurons from the raphe nuclei (Häring et al. 2007) and noradrenergic neurons from the locus coeruleus (Scavone et al. 2010) were already shown to express CB1. Though according to our estimation based on [3H]CP55,940 binding data, these represent only a few per cent of the entire hippocampal CB1 population, the existence of this CB1 expression, originating from extrahippocampal neurons, but located in the hippocampus, has to be considered when interpreting any functional analysis. In particular, basal forebrain cholinergic neurons arise from Dlx-expressing neurons (Bachy and Rétaux 2006), and in a lac-Z reporter mouse line using the same regulatory Dlx5/6 intergenic region (Stühmer et al. 2002) as present in the Dlx5/6-cre recombinase transgene (Monory et al. 2006) the septum is lacZ-positive. Consistently with this notion, septal area is devoid of CB1 mRNA expression in the GABA-CB1-KO mice (Monory et al. 2006).
As the overwhelming majority of hippocampal CB1 is on GABAergic neurons (Marsicano and Lutz 1999), immunohistochemistry and radioligand binding show only minimal, albeit significant, differences between Glu-CB1-KO and wild-type littermates. Nevertheless, previous studies using the same mouse models demonstrated the functional importance of ‘glutamatergic’ CB1 in several behavioural paradigms (Monory et al. 2006, 2007; Steiner et al. 2008; Lafenêtre et al. 2009; Jacob et al. 2009; Bellocchio et al. 2010; Häring et al. 2011; Metna-Laurent et al. 2012). In agreement with such in vivo data, this study shows that the small change in receptor expression induces considerable consequences on G protein signalling in the hippocampal formation.
In this study, we applied different techniques to estimate CB1 amount in hippocampi of Glu-CB1-KO and GABA-CB1-KO mice. Western blotting, radioligand binding and qPCR consistently showed that glutamatergic neurons contain about one quarter of all hippocampal CB1, while GABAergic neurons contain about three quarters of all hippocampal CB1.
The goal of our study was to understand more about how the information processing during CB1-dependent G protein signalling is different between inhibitory (GABAergic) and excitatory (glutamatergic) neurons. One obvious idea was that the signalling machinery responds to a certain ligand concentration with different sensitivity in the various neurons. Cell type-specific signalling (same agonist showing different intracellular effects through the same receptor in different cells) was already shown for CB1 (Peters and Scott 2009). However, contrary to our expectations, we found no difference in the ED50 values of [35S]GTPγS binding stimulated by two synthetic and two endocannabinoids in Glu-CB1-KO versus GABA-CB1-KO mice.
The Emax values, however, were significantly different in Glu-CB1-KO versus GABA-CB1-KO mice, indicating a higher amount of CB1-activated G proteins in glutamatergic cells. Quantitative analysis of G proteins in the mutant mice revealed that the relatively minor amount of receptor in glutamatergic cells is responsible for over 50% of the total cannabinoid-activated G proteins. Contrarily to what we observed in Glu-CB1-KO mice, hippocampal CB1 in GABAergic cells is responsible for only about 20–30% of the total cannabinoid-activated G proteins. In other words, signal amplification at the CB1-G protein interface is much higher in the glutamatergic cells than in the GABAergic cells. Taking into account that CB1 density is much higher in GABAergic cells than in glutamatergic cells, our data are in good agreement with the observation of Breivogel et al. (1997) that CB1-G protein coupling differs by region in the rat brain and the higher the receptor density in a particular region, the lower the receptor-G protein amplification factor is. We have now extended this observation to neuronal types as well.
The more effective CB1 signalling on glutamatergic cells is in good agreement with the observation that in the lateral amygdala, cannabinoid actions on glutamatergic synaptic transmission override those on GABAergic synaptic transmission, leading to an overall decrease of excitability (Azad et al. 2003). Moreover, according to behavioural studies (Bellocchio et al. 2010; Rey et al. 2012), systemic administration of low doses of cannabinoids activates CB1 on glutamatergic terminals, while high doses activate CB1 on GABAergic terminals. Somewhat conflicting these in vivo data, dose–response curves of cannabinoid-induced suppression of excitatory or inhibitory post-synaptic currents in hippocampal cultures revealed that the ED50 at glutamatergic synapses was approximately 30 times bigger than at GABAergic ones (Ohno-Shosaku et al. 2002). Electrophysiological measurements revealed another kind of difference between CB1 signalling in glutamatergic and GABAergic cells as well. These measurements showed that in glutamatergic cells CB1 has only a minimal (5–7%) tonic/constitutive activity (Roberto et al. 2010), while in GABAergic cells this value is 30–40% (Slanina and Schweitzer 2005). Therefore, the tonic and phasic activities of the endocannabinoid system seem to be unevenly distributed, CB1 in glutamatergic cells being in greater part responsible for the phasic activity than the receptor on GABAergic cells.
THC administration was shown to disrupt hippocampus-dependent memory formation through CB1 on GABAergic neurons (Puighermanal et al. 2009). Recently, it was shown that this memory-disturbing effect of THC is exacerbated by chronic caffeine (Panlilio et al. 2012) and this effect is mediated by the adenosine A1 receptor (Sousa et al. 2011). Interestingly, however, A1 receptor, though present in GABAergic neurons, does not directly participate in regulation of GABA release. Nevertheless, the study showed influence of A1 receptor activation on CB1-mediated decrease of GABA and glutamate release as well as CB1-mediated G-protein activation. Chronic caffeine treatment leads to an up-regulation of A1 and a down-regulation of CB1. Yet, THC-induced amnesic effects are intensified in caffeine-treated mice. As the study uses wild-type mice, it is not possible to tell, whether CB1 down-regulation occured in all hippocampal neurons or only in selected populations. These surprising results probably originate from a complex net effect of activation of different memory-related neuronal circuits.
An important limitation of experiments using [35S]GTPγS binding assays is that this approach has a strong bias towards pertussis toxin (PTX)-sensitive (Gi/Go) G proteins partly because PTX sensitive G proteins are more abundant and partly because they have a much faster guanine nucleotide exchange rate (Milligan 2003). Consequently, our experiments likely measured mainly CB1-dependent activation of Gi/Go proteins. Nevertheless, although the assay is not well suited for its detection, we cannot exclude the participation of other G proteins in the cannabinoid effects. Cannabinoid-induced activation of Gs (Glass and Felder 1997; Hampson et al. 2000; Bash et al. 2003; Peters and Scott 2009) and Gq proteins (Lauckner et al. 2005; McIntosh et al. 2007) were previously reported in in vitro systems. It is therefore possible that PTX-insensitive G proteins are responsible for part of the biphasic behavioural effects of cannabinoids.
Taken together, we describe here a neuron-type specific CB1-dependent G protein signalling in the mouse hippocampus. A far more effective receptor-G protein coupling in glutamatergic cells explains why this receptor population responds already to low doses of cannabinoids and why it also seems to be engaged in more physiological processes.