The object of this study was to characterize properties of nAChRs triggering catecholamine release in brain slice preparations of mice. We furthermore compared the two species, rats and mice, and the outflow following two types of stimulus, nicotine and electrical field stimulation. The principal findings of our study confirm and significantly extend previous observations made on mouse synaptosomes and indicate major species and regional differences of catecholamine outflow: (1) nicotine-induced [3H]noradrenaline outflow from hippocampal slices was significantly larger in rats than in mice. (2) Irrespective of species, electrical field stimulation of slices produced significantly more [3H]noradrenaline release from both the hippocampus and the neocortex than [3H]dopamine outflow from the striatum. (3) The nicotine-evoked catecholamine release was largely prevented in all slice preparations by the presence of TTX. (4) Nicotine-induced [3H]dopamine outflow from the striatum and [3H]noradrenaline release from the hippocampus of mice required the presence of the β2nAChR subunit. (5) Targeted deletion of the α5 subunit gene had no significant effect on [3H]dopamine outflow from the mouse striatum in response to nAChR activation.
The modulation [3H]noradrenaline outflow from hippocampal slices by nAChR activation was species dependent
In contrast, this ratio diminished to 0.13 in mouse hippocampal slices, indicating that nAChR activation triggers NA release less efficiently in the mouse than in the rat hippocampus. This observation is in line with a recent report showing that nicotine-evoked [3H]noradrenaline outflow from hippocampal synaptosomes is larger in rats than in mice (Azam and McIntosh, 2006). Such species difference has also been reported for the neocortex, where [3H]noradrenaline outflow induced by nicotine was significantly larger in human than in rat brain slice preparations (Amtage et al., 2004). Whether properties of these nAChRs in the human hippocampus compare better with the mouse than with the rat is not known. Species differences in nAChRs become critical though when applying observations from animal models to neuropathological mechanisms in men (Kaiser and Wonnacott, 2000; Picciotto et al., 2001; Amtage et al., 2004).
The impact of nAChR activation on catecholamine outflow differs between brain regions
Transmitter outflow in response to electrical field stimulation is the sum of several parameters, such as the number of presynaptic specializations, reuptake and degradation mechanisms and feedback inhibition by presynaptic autoreceptors (Valenta et al., 1988; Boehm and Huck, 1995). Any of these parameters might contribute to the larger [3H]noradrenaline release from neocortex and hippocampus compared to a smaller [3H]dopamine outflow from the striatum. The smaller effect of electrical field stimulation on [3H]dopamine outflow in the striatum did not go together with a reduction of nicotine effects. Hence, indices for nicotine-induced [3H]dopamine outflow set in relation to electrical field stimulation in mice yielded 0.93 (striatum), opposed to 0.13 (hippocampus, Table 1). These data, and the indices calculated for rat neocortical, hippocampal and striatal slices (0.21, 0.81 and 1.45, respectively, Table 1) suggest that the impact of nAChR activation on catecholamine release distinctly differs in these structures.
Indirect mechanisms of nAChR-modulated catecholamine release markedly outweigh direct mechanisms
Our finding that TTX inhibited nicotine-evoked catecholamine outflow in all our preparations by about 90% are in line with previous observations made on human and rat brain slice preparations (Sacaan et al., 1995; Marshall et al., 1996; Wonnacott, 1997; Lena et al., 1999; Leslie et al., 2002; Amtage et al., 2004; Barik and Wonnacott, 2006). Quite the opposite, nicotinic agonist-induced catecholamine release from rat synaptosomes is clearly less sensitive to an inhibition by TTX (Soliakov et al., 1995; Clarke and Reuben, 1996; Marshall et al., 1996; Leslie et al., 2002). To our knowledge, effects of TTX on nicotine-induced catecholamine release have not been studied in mouse synaptosomal preparations, though [86Rb+] efflux from mouse thalamic synaptosomes in response to nicotine was partly inhibited (by 42%) in the presence of TTX (Marks et al., 1995).
A sizeable effect of TTX indicates that a major part of nAChRs, though still on the catecholaminergic axon, is too far distant from active zones to trigger exocytosis directly by calcium influx through the nAChR channel pore (Wonnacott, 1997; Kristufek et al., 1999). As even synaptosomal preparations may show some TTX sensitivity (Soliakov et al., 1995; Marshall et al., 1996), nAChRs that trigger catecholamine release by a direct (TTX-resistant) and an indirect (TTX-sensitive) mechanism may coexist in relative close proximity (i.e., on isolated synaptosomes).
nAChRs modulating catecholamine outflow indirectly may in addition reside on glutamatergic axons, as shown in rat striatal slice preparations (Wonnacott et al., 2000; Kaiser and Wonnacott, 2000). Indirect effects of nAChR activation are more complex in the hippocampus, where both GABA-ergic and glutamatergic mechanisms are involved (Leslie et al., 2002; Barik and Wonnacott, 2006). In the rat, these indirect effects appear to be mediated by α7 nAChRs (Kaiser and Wonnacott, 2000; Wonnacott et al., 2000; Barik and Wonnacott, 2006).
Our experiments revealed that nicotine-evoked [3H]dopamine outflow from mouse striatal slices was reduced to 6% (compared to controls) in animals lacking the β2 nAChR subunit. These observations are in line with data obtained in striatal synaptosomal preparations, where β2 null mutations were found to eliminate nicotine-induced [3H]dopamine release (Whiteaker et al., 2000; Grady et al., 2002; Champtiaux et al., 2003; Salminen et al., 2004). However, our results imply that not only direct (TTX-resistant) but also indirect mechanisms depend on nAChRs containing the β2 subunit, as most of the [3H]dopamine outflow from mouse striatal slices in response to nicotine was owing to an indirect (TTX-sensitive) effect. nAChRs containing the β2 subunit also mediate the indirect (action potential-dependent) dopamine release caused by endogenous ACh in striatal slice preparations (Zhou et al., 2001).
We have shown that β2 null mutations greatly reduced catecholamine outflow not only from striatal but also from hippocampal slices. These data are in line with a recent study showing that [3H]noradrenaline outflow in response to nicotine is abolished in hippocampal synaptosomes prepared from β2-KO animals (Azam and McIntosh, 2006). By the combined use of subunit-specific conotoxins (α-MII, α-BuIA, α-PIA and α-AuIB) and KO animals (β2, β3, β4 and α4) the authors identified two types of nAChRs modulating [3H]noradrenaline outflow from synaptosomes: α6(α4)β2β3β4 and α6(α4)β2β3.
Pharmacological evidence suggests that receptors modulating [3H]noradrenaline outflow in rat hippocampal synaptosomes are distinct from those in mice and contain the subunits α3 and β4 (Clarke and Reuben, 1996; Luo et al., 1998; Azam and McIntosh, 2006). Hence, α-Ctx MII fully inhibits [3H]noradrenaline outflow in mice but is ineffective in rat synaptosomes. Likewise, α-Ctx PIA which reduces [3H]noradrenaline outflow by about 80% in mice potentiates nicotine effects in rat hippocampal synaptosomes (Azam and McIntosh, 2006)). The significantly larger outflow of [3H]noradrenaline in response to nicotine seen in rat compared to mouse hippocampal synaptosomes (Azam and McIntosh, 2006) may thus be caused by more efficient receptors, a higher number of receptors, or both.
Synaptosomes taken from adult mice do not display significant [3H]noradrenaline outflow above baseline (Azam and McIntosh, 2006). As most of our experiments were performed on hippocampal slices of 6–8 weeks old mice, nAChRs modulating [3H]noradrenaline release by indirect mechanisms not residing on noradrenergic projections may play a dominant role. The absence of nicotine-evoked [3H]noradrenaline outflow from hippocampal slices taken from β2-KO animals indicates that in mice, these indirect mechanisms crucially depend on the presence of the β2 subunit.
Targeted deletion of the nAChR α5 subunit gene
Three main types of hetero-oligomeric nAChRs have been identified in mouse dopamine terminal fields: α4β2*, α6β2* and α4α6β2*. Whereas α6-containing receptors are sensitive to α-CtxMII, those made of α4β2* are not (Champtiaux et al., 2003). α5 was present in 9% of purified α4* nAChRs but in only 1% of α6* receptors (Champtiaux et al., 2003). Nonetheless, targeted deletion of the α5 subunit gene affected not only α-CtxMII-insensitive (presumably mediated by α4β2* receptors) but also α-CtxMII-sensitive (presumably mediated by α6* receptors) ACh-induced outflow of [3H]DA, albeit in an opposite manner: Whereas maximally induced release (Rmax) in the presence of 100 nMα-CtxMII was reduced in the α5 KO, the Rmax of the α-CtxMII-sensitive component was enhanced (Salminen et al., 2004). The report makes no mention whether the two opposite effects of the α5-KO level off when looking at overall outflow in the absence of α-CtxMII.
As in synaptosomal preparations (Kulak et al., 1997; Kaiser et al., 1998; Kaiser and Wonnacott, 2000; Grady et al., 2002; Champtiaux et al., 2003; Salminen et al., 2004) we found α-CtxMII to inhibit [3H]dopamine release from mouse striatal slices by about 50% in response to nicotine. Our assay is, however, not sufficiently robust to distinguish possible effects of a deletion of the α5 subunit on the α-CtxMII-sensitive and the α-CtxMII-insensitive component of [3H]dopamine release, as carried out in mouse striatal synaptosomes (Salminen et al., 2004). The overall release (Rmax and EC50 values in the absence of α-CtxMII) induced by three nicotinic agonists nicotine, cytisine and DMPP was not significantly affected in mice lacking the α5 subunit. These results are in contrast to observations we made in cultured sympathetic neurons, where targeted deletion of the α5 subunit gene caused a large enhancement of nicotine-evoked [3H]noradrenaline outflow (Fischer et al., 2005).
Impact of nAChRs located in the projection areas of catecholaminergic neurons
Excitation of dopaminergic neurons in the midbrain and of noradrenergic neurons in the locus coeruleus play a major, if not dominant, role in the release of catecholamines upon systemic application of nicotine (Mitchell, 1993; Fu et al., 1998; Mansvelder and McGehee, 2000; Picciotto and Corrigall, 2002; Mansvelder et al., 2003; David et al., 2006). The collective activation of projecting axons by electrical field stimulation in brain slices provides some indirect evidence on the overall release capacity of these systems. When set in relation to the electrically induced outflow, nicotine by acting on receptors in the terminal fields is about equi-effective in stimulating [3H]dopamine release from the striatum, but significantly less so in evoking [3H]noradrenaline outflow from the neocortex of both rats and mice. The modulation of [3H]noradrenaline outflow in the hippocampus by nicotine is species-dependent: sizeable in rat, but small in the mouse hippocampal slice preparation.
Our comparative observations made in striatal, hippocampal and neocortical slice preparations from rats and mice showed regional and species dependent differences of nAChR properties in the terminal fields of catecholaminergic projections. In mice, nicotine-evoked catecholamine outflow is primarily mediated by action potentials and dependent on nAChRs containing the β2 subunit.