Criterios y Procedimiento

Early autoradiographic studies indicate that adenosine receptors are highly localised in some areas of the brain. Incubation of rat brain sections with the radiolabelled A1R agonist [3H] cyclohexyladenosine produces A1R labelling throughout the brain

particularly in the cerebellum where dense staining is observed in the molecular layer, moderate staining in the granule layer and very light staining in the white matter. In brain sections from reeler mice with distorted cerebella the [3H] cyclohexyladenosine labelling is distorted in a pattern corresponding to the parallel fibres of the granule cells providing evidence for the specific localisation of A1R to these axons within the

molecular layer (Goodman and Snyder 1982).

Immunohistochemical detection of A1R using polyclonal antisera against identical

peptides from the rat and human A1R provides the resolution to individual processes

and cell bodies within the cerebellum not attained with autoradiography. The A1R

staining intensity within the molecular layer is not as intense with this detection method as that seen with autoradiography which may be due to the protocol causing degeneration of the many small unmyelinated axons and dendrites, or A1R present on

them, in this area of the brain. Immunohistochemical staining reveals moderate labelling of granule cells in the granule layer and heavy labelling of basket cells that often envelop the lightly-labelled Purkinje cells in the molecular layer of the cerebellum suggesting the possibility that A1R may modulate interactions between

Double immunolabelling shows co-localisation of the A1R and P2Y1receptor in the

Purkinje cell and molecular cell layers of the rat cerebellum. Co- immunoprecipitation of the the A1R with the P2Y1 receptor using an A1R antibody

confirms that the receptors interact as hetero-oligomers and are present in the absence of any receptor activation by exogenous agonists so may be functionally significant (Yoshioka, Hosoda et al. 2002).

Immunoprecipitation of rat cerebellum synaptosomes reveals areas where

metabotropic glutamate type 1α receptors (mGlu1αR) and A1R do not co-distribute

and areas where the two receptors interact to form heteromers dependent on the mGlu1αR C-terminal tail. At the level of calcium mobilisation in response to

application of adenosine and glutamate agonists this interaction shows a synergistic effect and therefore functional significance in transfected HEK293 cells (Ciruela, Casado et al. 1995).

A number of studies describe A2AR expression in dopamine-innervated areas of the

rat brain but have provided little evidence for the presence of A2AR outside of the

striatum. A subpopulation of Purkinje cells within the rat cerebellum show a low expression of A2AR when incubated with a 35S-labelled cRNA probe for the A2AR

(Svenningsson, Le Moine et al. 1997). Immunohistochemistry using a high affinity monoclonal antibody specific to the A2AR shows a light labelling of scattered

Purkinje cells in the rat cerebellum correlating with low A2AR mRNA levels whilst

also confirming localisation of A2AR mainly to areas of the brain receiving

dopaminergic projections (Rosin, Robeva et al. 1998). Positron emission tomography (PET) scans using the A2AR antagonist [11C]TMSX demonstrate that

A2AR are enriched in the human striatum and are widespread in other areas of the

human brain with moderate levels of A2AR binding in the cerebellum (Mishina,

Ishiwata et al. 2007).

Minimal support exists for the presence of A2BR within the cerebellum although a

functional role has been suggested. Adenosine can synergistically potentiate the rapid [Ca2+]i transient increase evoked by the action of ATP at P2Y receptors in the

majority of cerebellar astrocytes. This effect is mimicked by the A1R and A2A/BR

agonist NECA but not prevented by DPCPX, an antagonist at A1R and A2AR. A

similar number of the cerebellar astrocytes positively stain for A2BR antibody

(Jimenez, Castro et al. 1999). Application of ADA to eliminate endogenous adenosine activity in cultured cerebellar granule cells either increases or decreases [Ca2+]i depending on the cell state suggesting a role for multiple adenosine receptors

in the same cell type. The effects of various agonists and antagonists at adenosine receptors and the identification of adenosine receptor subtype cDNA in the cerebellar granule cells indicates the possible involvement of A1R, A2BR and possibly A3R in

calcium modulation (Vacas, Fernandez et al. 2003).

Few studies have demonstrated the presence or role for A3R in the cerebellum. The

radioligand [125I] AB-MECA binds specifically to A1R and A3R in mouse brain

membrane preparations and the majority of binding is displaced by the A1R

antagonist CPX leaving low levels of residual binding representing A3R. In the

mouse brain the highest levels of residual radioligand binding are localised in the cerebellum and correspond with A3R mRNA detection although these levels are very

transgenic mice containing a construct with a promoter region of the A3R gene coupled to the β-galactosidase reporter gene reveals β-galactosidase activity in the