Adenosine A 2A receptors (A 2A R)

dopamine D₁ receptors (D₁R) (Ferré et al., 1996; Fuxe et al., 1998), providing yet

another mechanism from which A₁R can influence neuronal activity.

A₁R are coupled with G_i/o, inhibiting the adenylate cyclase and decreasing cyclic adenosine monophosphate (cAMP) (van Calker et al., 1979) that activates potassium channels, blocks calcium channels and increases inositol trisphosphate (IP₃) levels (Fredholm et al., 2001; Rogel et al., 2005; Tawfik et al., 2005). Consequently, A₁R activation modulates neuronal activity by inhibiting neurotransmitter release and reducing the firing rate. The most evident effect of A₁R activation in neuronal circuits of adult mammals is the selective depression of excitatory transmission (Dunwiddie and Haas, 1985).

Some of the roles of A₁R were unveiled through Adora1-KO (A₁R KO) mice. Two constitutive, global A₁R KO mice lines have been generated (Johansson et al., 2001;

Sun et al., 2001; Fedele et al., 2006), as well as a brain-specific conditional A₁R KO mice (Bjorness et al., 2009) using the Cre/loxP strategy in which the Cre transgene expression was placed under the control of the alpha-Ca²⁺

/calmodulin-dependent protein kinase II (CaMKII-α) promoter to provide both regional and temporal specificity of Cre expression and thus of A1R gene (Tsien et al., 1996).

Focal deletion of A1R in hippocampal CA1 or CA3 neurons has been attained by local injection of adeno-associated virus (AAV) vectors containing the Cre transgene construct into those brain areas of mice with a critical exon of Adora1 flanked by loxP sites (Scammell et al., 2003), which also allowed a temporal and regional specificity of A₁R deletion.

1.2. Adenosine A2A receptors (A2AR)

High levels of the A_2AR are found in particular regions of the brain, namely in the dorsal and ventral striatum, as well as in the olfactory tubercle (Schiffmann et al., 1991b;

1

Fink et al., 1992; Dixon et al., 1996; Svenningsson et al., 1997a; 1997b; Rosin et al., 1998). Despite that, it is recognized that A_2AR are also present throughout the brain, albeit with a considerably lower density, namely in the hippocampus and cortex (Dixon et al., 1996; Svenningsson et al., 1997a). In addition, A_2AR show different subregional and cellular expression patterns within a specific tissue. For example, in the striatum, A_2AR are mainly localized in the postsynaptic striatopallidal MSNs of the indirect pathway (Schiffmann et al., 1991a; Svenningsson et al., 1999b; Rebola et al., 2005a), where they interact with dopamine D₂ receptors (D₂R) (Ferré, 1997; Ferré et al., 1997; Hillion et al., 2002). A_2AR are also detected at lower levels at presynaptic sites in cortico-striatal terminals (Popoli et al., 2002; Martire et al., 2010) and in the hippocampus (Rebola et al., 2005a), where they are also present postsynaptically (Rebola et al., 2008). In addition to the synaptic enrichment, A_2AR are also located in astrocytes (Li et al., 2001;

Nishizaki et al., 2002; Matos et al., 2012b), microglia (Fiebich et al., 1996; Küst et al., 1999) and in endothelial cells of brain capillaries, where they play an important role controlling brain vascular function (O'Regan, 2005; Mills et al., 2011).

In the brain A2AR are coupled with Gs/olf, stimulating the adenylate cyclase and increasing cAMP (Fredholm et al., 2000; Kull et al., 2000; Corvol et al., 2001; Hervé et al., 2001). A2AR signaling is classically described as occurring via a protein kinase A (PKA)-dependent pathway, though A2AR signaling through a protein kinase C (PKC)-dependent pathway in hippocampal synaptosomes has also been demonstrated (Gubitz et al., 1996; Nörenberg et al., 1998; Cunha and Ribeiro, 2000a; 2000b; Queiroz et al., 2003; Rebola et al., 2003b; Pinto-Duarte et al., 2005). Interestingly, A_2AR seem to have limited   impact   on   the   control   of   ‘basal’   synaptic   transmission   but   play   a   crucial   role in

controlling synaptic plasticity (Cunha, 2008a).

In addition, the scope of action and effects of A_2AR manipulation, including the triggered signaling pathways, should be evaluated together with their ability to

1 heteromerize with different other G protein-coupled receptors, such as A₁R (O'Kane and

Stone, 1998; Ribeiro, 1999; Ciruela et al., 2006a; 2006b), D₂R (Ferré, 1997; Ferré et al., 1997; Hillion et al., 2002), metabotropic glutamate type 5 receptors (mGluR₅) (Ferré et al., 2002; Tebano et al., 2005), N-methyl-D-aspartate receptors (NMDAR) (Nörenberg et al., 1998; Ribeiro, 1999), and cannabinoid CB₁ receptors (CB₁R) (Carriba et al., 2007;

Ferré, 2007; Ferré et al., 2007; Tebano et al., 2009).

Some of the A_2AR functions were also unveiled through Adora2A-KO (A_2AR KO) mice. Four constitutive, global A_2AR knockout mouse lines from different genetic backgrounds have been generated (Ledent et al., 1997; Chen et al., 1999; Day et al., 2003; Huang et al., 2006), as well as two different brain-regional deletion of A_2AR: in the forebrain (i.e., striatum, cortex, hippocampus) (Bastia et al., 2005; Xiao et al., 2006) or striatum (Shen et al., 2008; Yu et al., 2009), using the Cre/loxP strategy in which Cre transgene expression was placed under the control of the forebrain neuron-specific CaMKII-α or Dlx5/6 promoter, respectively.

1.2.1. Hippocampal A2AR

In the hippocampus A2AR are mainly expressed in neurons (Rebola et al., 2003b;

2005a; 2005b), being enriched in the active zone of presynaptic terminals (Rebola et al., 2005a; 2005b), where they control the release of glutamate (Cunha and Ribeiro, 2000a), acetylcholine (Cunha et al., 1994b; 1995b; Jin and Fredholm, 1997; Rebola et al., 2002) and serotonin (Okada et al., 2001), among others (Sebastião and Ribeiro, 1996).

However, hippocampal A_2AR is particularly enriched in glutamatergic synapses (Rebola et al., 2005b), where these receptors play a tight control on the release of glutamate (Cunha and Ribeiro, 2000a; Lopes et al., 2002). The final target of A_2AR modulation in nerve terminals seems to be calcium channels (Mogul et al., 1993; Umemiya and Berger, 1994; Gubitz et al., 1996; Gonçalves et al., 1997). Postsynaptic hippocampal

1

A_2AR are able to modulate plasticity, being required for long-term potentiation (LTP) (Rebola et al., 2008; Fontinha et al., 2009; Diógenes et al., 2011; Dias et al., 2012), where blockade of these receptors impaired the response to conditioned behaviors (Fontinha et al., 2009). Hippocampal A_2AR are also present in astrocytes (Nishizaki et al., 2002), where they control glutamate release and uptake through modulating the activity of glutamate transporters (Nishizaki et al., 2002; Matos et al., 2012b; Matos et al., 2013).

The overall control of extracellular glutamate levels (derived from neurons or astrocytes) and LTP by hippocampal A_2AR, may play crucial roles in physiological and noxious conditions affecting the hippocampal functions, like memory and epilepsy.

Actually, several reports observed increased A_2AR density in the hippocampus upon brain harmful conditions (Cunha et al., 1995a; Rebola et al., 2003b; Cunha, 2005; Cunha et al., 2006; Duarte et al., 2006; Canas et al., 2009a), namely in epilepsy (Doriat et al., 1999; Cognato et al., 2010), which seems to be responsible for an enhanced facilitation of glutamatergic synaptic transmission (Rebola et al., 2003b; Costenla et al., 2011) and acetylcholine release (Lopes et al., 1999a; 1999b).

In addition, it was shown that high frequency of neuronal firing in the hippocampus, leads to ATP release and a preferential activation of A2AR (Cunha et al., 1996a; 1996b;

Cunha, 2005), which seems to be able to attenuate A1R function (Cunha et al., 1994a;

Lopes et al., 2002; Pinto-Duarte et al., 2005). In agreement, the percentage of nerve terminals with A_2AR in the hippocampus that are A₁R-positive is around 80% (Rebola et al., 2005b). Consequently, A_2AR have a major role in the control of A₁R functions, probably through intracellular transducing systems (Lopes et al., 1999a; 1999b) or maybe through receptors dimerization (Ciruela et al., 2006b).

1 Hippocampal A_2AR are also able to transactivate TrkB receptors in the absence of

the ligand (Lee and Chao, 2001), being required for normal BDNF levels and functions in the hippocampus (Diógenes et al., 2004; 2007; Tebano et al., 2008).