To assay for elevated receptor expression across different brain structures, we performed autoradiographic radioligand binding assays

with iodinated epibatidine, which mainly binds α4β2∗ and α3β4∗ nAChRs (Perry CHIR-99021 cost and Kellar, 1995) (Figures 5A and 5B). Competition with cold cytisine, which binds with higher affinity to α4β2∗ than to α3β4∗ receptors (Marks et al., 2010), was done to distinguish α3β4∗ from overlapping α4β2∗ binding sites (Zoli et al., 1998) (Figures 5C and 5D). In WT mice, discrete brain regions resistant to cytisine competition labeled well-known α3β4∗ sites such as MHb, IPN, and superior colliculus (Figure 5C). In Tabac mice, increased radioligand binding to cytisine-resistant sites was detected in these areas and in additional brain structures, including the VTA, SuM, substantia

nigra, and striatum (Figure 5D). A strong correlation between radioligand signal and eGFP fluorescence was detected in all analyzed CNS structures (Figure 5E and Table S1). Densitometric analyses indicated significantly increased cytisine-resistant signals in α3β4∗-expressing regions in Tabac mice (Table S1), while α4β2∗ epibatidine binding sites such as cortex and thalamus did not differ between control and Tabac mice (Figures 5A and 5B), indicating that elevated surface receptors are present in sites corresponding to endogenous β4 expression sites. To exclude the possibility that the increased radioligand signal could reflect increased cell number, we quantified the cell density in MHb of Tabac and WT mice and observed no significant http://www.selleckchem.com/products/ch5424802.html differences (Figure S3). These data show that the enhanced nicotine-evoked currents in Tabac mice result from β4-mediated recruitment of additional functional α3β4∗ nAChR complexes on the cell surface. Taken together, the anatomic mapping and ISH results presented in Figure 3 and the receptor binding assays presented in Figure 5 provide compelling evidence that α3β4∗ nAChRs are located both on the cell soma and in the axon termini. For example, the relatively

light staining of the IPN by ISH in Tabac mice strongly suggests that the very heavy expression of functional α3β4∗ receptors, detected in this structure by receptor binding, results because from both local synthesis in the IPN and the presence of receptors synthesized in the MHb and transported to presynaptic termini in the IPN. This is consistent with the well-documented effects of presynaptic nAChRs on synaptic release and neurotransmission (McGehee et al., 1995), and suggests that Tabac mice will be an important tool for further dissection of the roles of presynaptically and postsynaptically expressed nAChRs. We were next interested in the effects of elevated nAChR expression on the behavioral responses of Tabac mice to nicotine.