Using in vitro electrophysiological recordings, this study compared the ability o f DA
D] receptors to inhibit firing o f DAergic neurons within the VTA o f wildtype and
adenosine A2A receptor knockout mice. No difference in the baseline firing frequency
in the wildtype compared to the adenosine Aja receptor knockout mice was observed,
which suggests that in the absence o f adenosine A2A receptors, the cell physiology
was unaltered.
al. (1997) and Centonze et al. (2002) also previously reported that the application o f 0.1-30 pM quinpirole for 3-5 min caused a reversible inhibition o f firing activity on
the DAergic neurons o f the VTA in inbred mouse strains. In addition, Bowery et al.
(1994) demonstrated that in rats the cumulative addition o f 0.003-0.1 pM quinpirole
caused inhibition in firing in DAergic neurons in the VTA with almost complete
inhibition at 0.1 pM. Microdialysis studies involving the perfusion o f quinpirole into
the VTA show a reduction in the efflux o f DA and in VTA DA cell firing, which
suggests that DA D] receptors inhibit the local release o f DA (Kalivas et al. 1989;
Chen et al. 2000b). The specific involvement o f the DA D% receptors in the VTA was
confirmed by the addition o f the DA D2 receptor antagonist, sulpiride, which reversed
the inhibitory effect o f quinpirole in VTA DA cells (Fig. 2.5), as shown by others
(Bemardini et al. 1991; Bowery et al. 1994; Mercuri et al. 1997; Centonze et al.
2002).
In the adenosine A2A receptor mice, however, the firing frequency o f VTA DA
neurons was not reduced to the same extent as shovm in wildtype mice, even when the
concentration o f quinpirole was increased to 50 pM (Fig. 2.3 and 2.4). A significant
genotype effect was revealed upon comparison o f the percentage inhibition in
response to quinpirole in the wildtype and adenosine A2A receptor knockout mice
(Fig. 2.4). A decrease in the maximal response was seen in the adenosine A2A receptor
knockout mice compared to wildtype mice although there was no change in EC50 (Fig.
2.4).
In more recent unpublished studies from our research group the effect o f single rather
firing in the VTA in wildtype and adenosine A2A receptor knockout mice, creating a
concentration response curve, have been investigated. In these experiments both
genotypes showed a similar maximal reduction in DA cell firing in the VTA in
comparison to the reduced maximal response in the inhibition o f VTA DA cell firing
(shown here) following cumulative applications in the adenosine A2A receptor
knockout mice. During the single applications o f quinpirole the brain sections were
exposed to quinpirole for 15 min whereas during the cumulative concentration
response, presented here, the brain sections were exposed to each concentration o f
quinpirole for 5 min before the addition o f the next concentration. The concentration-
response curve following individual dosing (not shown here) shifted to the left; an
increase in the inhibition o f DAergic firing was seen at lower doses (0.001-1 pM,
EC$o=9 nM) in comparison to cumulative dosing (0.01-30 pM, EC5o=0.04 pM),
which suggests that perhaps cumulative dosing did not allow for a full effect to be
reached at each concentration. These results suggest that the extent o f quinpirole-
induced inhibition o f DA cell firing may be time dependent. Bemardini et al. (1991)
looked at various strains o f wildtype inbred mice and also demonstrated that
individual dosing o f lower concentrations o f quinpirole (0.001-1 pM, EC$o=9nM)
resulted in complete inhibition in DAergic firing in the VTA. In the individual dose
study the inhibition in DAergic firing was reduced in adenosine A2A receptor
knockout mice, with a significant difference achieved at 3 nM.
The findings from this study suggest a possible alteration in the behaviour o f the DA
D2 receptors in the VTA in adenosine A2A receptor knockout mice. However, the
hypothesised alteration in DA D2 receptor function cannot be directly attributed to the
was no functional evidence for adenosine A%A receptors in the VTA, shown in this
study and through autoradiographic binding studies and immuohistochemistry
(Sebastiao et al. 1996; Moreau et al. 1999). Therefore, altered DA D2 receptor
function may be due to adaptive changes in adenosine A2A receptor knockout mice.
There is evidence to support the presence o f heteromeric complexes containing
adenosine A2A and metabotropic glutamate 5 receptors (mGlu5), as well as adenosine
A2A and DA D2 receptors, in striatal neurons (Ferre et al. 2002; Fuxe et al. 2003). It
has been shown that adenosine A2A and DA D2 receptors may play a major role in
mGlu5 receptor modulation o f striatal function (Popoli et al. 2001; Nishi et al. 2003;
Domenici et al. 2004; Rodrigues et al. 2005), which is likely to be disrupted in
adenosine A2A receptor knockout mice. Moreover, electrophysiological experiments
have also provided evidence that the dendritic release o f DA may regulate the firing
rate o f DAergic neurons in the VTA through DA D2 receptors located in
glutamatergic terminals (Koga et al. 2000) which may be altered in adenosine A2A
receptor knockout mice.
It has also been postulated that the somatodendritic release o f DA in the VTA
modulates cell firing by the interaction with local DA D2 receptors (Adell et al. 2004).
However, endogenous DA, via DA D2 receptors, does not appear to have an effect on
the firing rate o f DAergic neurons in the VTA as no change in the firing frequency o f
DAergic neurons was observed following exposure to the DA D2 antagonist, sulpiride
The mechanism underlying the alteration in DA D] receptor function in adenosine
A2A receptor knockout mice is still unclear. Neuronal circuitries are absent in such in
vitro studies therefore the potential influence o f neuronal circuitries is absent. The
absence o f adenosine A2A receptors may influence the GABAergic feedback loop o f
the mesolimbic pathway, projecting from the NAc to the VTA. Further studies would