Immunohistochemical localization of adenosine A2A receptors in the rat central nervous system

Abstract

The A2A adenosine receptor (A2A-AR) transcript and radioligand binding sites have a distinct distribution in rat brain, restricted primarily to the striatum, nucleus accumbens and olfactory tubercles. We describe here the use of purified recombinant human A2A-ARs to generate a monoclonal antibody that has been used to better resolve the distribution of A2A-ARs in rat brain. The antibody can detect 1 ng of purified recombinant receptor by Western blotting and is potent (EC50 = 0.62 microg/ml) and highly selective for the A2A-AR subtype. By Western blotting, the apparent molecular mass of recombinant and rat striatal receptors shifts upon deglycosylation from 43-48 to 42 kilodaltons. Analyses of chimeric A1/A2A-ARs and synthesis of a blocking peptide pinpointed the epitope (SQPLPGER) of the antibody to the center of the third intracellular loop of the receptor. Incubation of rat striatal membranes with antibody reduces receptor coupling to G-proteins. In rat brain, dense A2A-AR-like immunoreactivity that is eliminated by the blocking peptide was found in the neuropil of the striatum, nucleus accumbens (rostral pole, core and shell), cell bridges of the striatum, olfactory tubercles, and areas of extended amygdala with somewhat lighter labeling in the globus pallidus and nucleus of the solitary tract. Light perikaryal labeling was found in other areas of the brain, including the cortex, hippocampus, thalamus, cerebellum, and portions of the hindbrain. The observed distribution of A2A-AR immunoreactivity throughout the neuraxis is consistent with the receptors' role in modulating dopaminergic neurotransmission and central control of cardiovascular function.

Increased desensitization of dopamine D2 receptor-mediated response in the ventral tegmental area in the absence of adenosine A2A receptors

“…(Ferre et al, 2008) and their heterodimers also have the potential to internalise differentially from each homomers and, thereby, desensitise differentially (Hillion et al, 2002;Torvinen et al, 2005;Vidi et al, 2008). We also show here that the A 2A receptor agonist, CGS21680, did not cause any pharmacological effect on the firing rate or pattern of dopamine neurons, confirming the lack of pharmacologically accessible A 2A receptors on dopamine neurons, which is in agreement with a low level of A 2A receptor expression in the VTA (Rosin et al, 1998;Rosin et al, 2003). CGS21680 also failed to acutely alter D 2 receptor function in the VTA, indicating a lack of direct pharmacological interaction between D 2 and A 2A receptors in the VTA, like those in the striatum (Azdad et al, 2009).…”

“…Adenosine A 2A receptors are G s/olf -coupled receptors for the endogenous neuromodulator adenosine (Fredholm et al, 2011). The expression of A 2A receptors in the brain is at the highest density in the soma of the GABAergic medium spiny neurons in the striatum, with significantly lower levels of expression elsewhere in the brain (Rosin et al, 1998;Svenningsson et al, 1999;Rosin et al, 2003). In the striatum, A 2A receptors co-localize with dopamine D 2 receptors (Ferre et al, 2008).…”

Adenosine A2A receptors in ventral striatum, hypothalamus and nociceptive circuitry

“…However, there is some evidence that both the dorsal and ventral striatum are directly accessed by nociceptive neurones (Newman et al, 1996) and that the ventral striatum, like many parts of the limbic system, modulates pain processing (see Millan, 1999). Using immunohistochemical techniques, most of the receptor expression is also seen in the basal ganglia (Rosin et al, 1998). However, it should be stressed that A 2A receptors are found at relatively low levels in somatosensory cortex (Johansson et al, 1997;Kelly et al, 2004) and their presence in the cortex has been confirmed by western blotting (Lopes et al, 2004), and thus a potential role of A 2A receptors in the central integration of pain cannot be completely ignored.…”

“…A 2A receptor antagonists show promising results as an adjuvant to L-DOPA therapy (see 2.3.). The dorsal striatum, which contains a high density of A 2A receptors (Rosin et al, 1998), appears to be the site of action for the antiparkinsonian effects of A 2A antagonists. The dorsal striatum receives its dopaminergic input from the substantia nigra (pars compacta), the predominant area of neurodegeneration in Parkinson's disease (Hirsch et al, 1988;Rinne, 1993).…”

“…Although the striatum contains the highest density of A 2A receptors in the brain (Rosin et al, 1998), morphological and functional studies have identified lower levels of A 2A receptors in other brain areas. A 2A receptors appear to be involved in some adenosine-regulated brain functions previously thought to be mediated solely by A 1 receptors.…”