Acetylcholine release stimulated by nicotinic agonists was measured as radioactivity released from perfused synaptosomes prepared from mouse interpeduncular nucleus (IPN) that had been loaded with [3 H]choline. Agonist-stimulated release was dependent upon external calcium and over 90% of released radioactivity was acetylcholine. The release process was characterized by dose response curves for 13 agonists and inhibition curves for six antagonists. a-Conotoxin MII did not inhibit this release, while a-conotoxin AuIB inhibited 50% of agonist-stimulated release. Comparison of this process with [ 3 H]dopamine release from mouse striatal synaptosomes indicated that different forms of nicotinic acetylcholine receptors (nAChRs) may mediate these processes. This was con®rmed by assays using mice homozygous for the b2 subunit null mutation. The deletion of the b2 subunit had no effect on agonist-stimulated acetylcholine release, but abolished agonist-stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the b2 subunit null mutation showed decreased dopamine release evoked by L-nicotine with no apparent change in EC 50 value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.

ABSTRACT␣4 and ␤2 nicotinic cholinergic receptor (nAChR) subunits can assemble in heterologous expression systems as pentameric receptors with different subunit stoichiometries that exhibit differential sensitivity to activation by acetylcholine, yielding biphasic concentration-effect curves. nAChR-mediated 86 Rb ϩ efflux in mouse brain synaptosomes also displays biphasic acetylcholine (ACh) concentration-response curves. Both phases are mediated primarily by ␣4␤2*-nAChR, because deletion of either the ␣4 or ␤2 subunit reduces response at least 90%. A relatively larger decrease in the component of 86 Rb ϩ efflux with lower ACh sensitivity occurred with partial deletion of ␣4 (␣4 ϩ/Ϫ ), whereas a larger decrease in the component with higher ACh sensitivity was elicited by partial deletion of ␤2 (␤2 ϩ/Ϫ ). Immunoprecipitation with selective antibodies demonstrated that more than 70% of [ 3 H]epibatidine binding sites in both regions contained only ␣4 and ␤2 subunits. Subsequently, ␣4 and ␤2 subunit content in the cortex and thalamus of ␣4 and ␤2 wild types and heterozygotes was analyzed with Western blots. Partial deletion of ␣4 decreased and partial deletion of ␤2 increased the relative proportion of the ␣4 subunit in assembled receptors. Although these methods do not allow exact identification of stoichiometry of the subtypes present in wild-type cortex and thalamus, they do demonstrate that cortical and thalamic nAChRs of the ␣4 ϩ/Ϫ and ␤2 ϩ/Ϫ genotypes differ in relative expression of ␣4 and ␤2 subunits a result that corresponds to the relative functional changes observed after partial gene deletion. These results strongly suggest that ␣4␤2-nAChR with different stoichiometry are expressed in native tissue.

[3H]Epibatidine binds to nAChR subtypes in mouse brain with higher (KD approximately 0.02 nM) and lower affinity (KD approximately 7 nM), which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of alpha7, beta2 or beta4 subunits. Most, but not all, higher and lower affinity binding sites require beta2 (Marks, M.J., Whiteaker, P., Collins, A.C., 2006. Deletion of the alpha7, beta2 or beta4 nicotinic receptor subunit genes identifies highly expressed subtypes with relatively low affinity for [3H]epibatidine. Mol. Pharmacol. 70, 947-959). Effects of functional alpha4 gene deletion are reported here. Deletion of alpha4 virtually eliminated cytisine-sensitive, higher-affinity [3H]epibatidine binding as did beta2 deletion, confirming that these sites are alpha4beta2*-nAChR. Cytisine-resistant, higher-affinity [3H]epibatidine binding sites are diverse and some of these sites require alpha4 expression. Lower affinity [3H]epibatidine binding sites are also heterogeneous and can be subdivided into alpha-bungarotoxin-sensitive and -resistant components. Deleting alpha4 did not affect the alpha-bungarotoxin-sensitive component, but markedly reduced the alpha-bungarotoxin-resistant component. This effect was similar, but not quite identical, to the effect of beta2 deletion. This provides the first evidence that lower-affinity epibatidine binding sites in the brain require expression of alpha4 subunits. The effects of alpha4 gene targeting on receptor function were measured using a 86Rb+ efflux assay. Concentration-effect curves for ACh-stimulated 86Rb+ efflux are biphasic (EC50 values=3.3 microM and 300 microM). Targeting alpha4 produced substantial gene-dose dependent reductions in both phases in whole brain and in most of the 14 brain regions assayed. These effects are very similar to those following deletion of beta2. Thus, alpha4beta2*-nAChRs mediate a significant fraction of both phases of ACh stimulated 86Rb+ efflux.

The nicotinic acetylcholine receptors (nAChR) assembled from α4 and β2 subunits are the most densely expressed subtype in the brain. Concentration-effect curves for agonist activation of α4β2*-nAChR are biphasic. This biphasic agonist sensitivity is ascribed to differences in subunit stoichiometry. The studies described here evaluated desensitization elicited by low concentrations of epibatidine, nicotine, cytisine or methylcarbachol of brain α4β2-nAChR function measured with acetylcholine stimulated 86 Rb + efflux from mouse thalamic synaptosomes. Each agonist elicited concentration-dependent desensitization. The agonists differed in potency. However, IC 50 values for each agonist for desensitization of 86 Rb + efflux both with high (EC 50 ≈3 μM) and low (EC 50 ≈ 150 μM) acetylcholine sensitivity were not significantly different. Concentrations required to elicit desensitization were higher that their respective K D values for receptor binding. Even though the two components of α4β2*-nAChR mediated 86 Rb + efflux from mouse brain differ markedly in EC 50 values for agonist activation, they are equally sensitive to desensitization by exposure to low agonist concentrations. Mice were also chronically treated with nicotine by continuous infusion of 0, 0.5 or 4.0 mg/kg/hr and desensitization induced by nicotine was evaluated. Consistent with previous results, chronic nicotine treatment increased the density of epibatidine binding sites. Acute exposure to nicotine also elicited concentration-dependent desensitization of both high sensitivity and low sensitivity acetylcholine-stimulated 86 Rb + efflux from cortical and thalamic synaptosomes. Although chronic nicotine treatment reduced maximal 86 Rb + efflux from thalamus, IC 50 values in both brain regions were unaffected by chronic nicotine treatment.

Cold-chain requirements affect worldwide distribution of many vaccines. In addition, vaccines requiring multiple doses impose logistical and financial burdens, as well as patient compliance barriers. To address such limitations, we have developed new technologies to prepare thermostable, single-shot, prime-boost microparticle vaccines. Antigen/adjuvant formulations containing glass-forming polymers and trehalose first are spray-dried to form glassy microparticles that confer thermostability. Atomic layer deposition (ALD) reactions conducted in fluidized beds are then used to coat the microparticles with defined numbers of molecular layers of alumina that modulate the timed release of the internalized antigen and act as adjuvants. We have used a model HPV16 L1 capsomere antigen to evaluate the properties of these technologies. Thermostabilized powders containing HPV16 L1 capsomeres were prepared by spray-drying, coated by ALD with up to 500 molecular layers of alumina, and injected into mice. Antigen distribution was assessed by live-animal IR dye tracking of injected labeled antigen. Antibody responses were measured weekly by ELISA, and neutralizing antibodies were measured by pseudovirus neutralization assays at selected time points. Thermostability was evaluated by measuring antibody responses after incubating ALD-coated antigen powders for one month at 50 °C. Single doses of the ALD-coated vaccine formulations elicited a prime-boost immune response, and produced neutralizing responses and antibody titers that were equivalent or superior to conventional prime-boost doses of liquid formulations. Antibody titers were unaffected by month-long incubation of the formulations at 50 °C. Single-dose, thermostable antigen preparations may overcome current limitations in HPV vaccine delivery as well as being widely applicable to other antigens.