Native nAChR-mediated whole-cell current responses. An identified MS/DB cholinergic neuron (no hyperpolarization-induced current, I_h) exhibited α7-nAChR-like current responses to 1 mM ACh and 10 mM choline (sensitive to blockade by 1 nM methyllycaconitine; MLA) but not to 0.1 mM RJR-2403, an agonist selective for α4β2-nAChRs (A), whereas an identified VTA DAergic neuron (evident I_h) showed both α7-nAChR-like (i.e., choline and MLA-sensitive components) and α4β2-nAChR-like (i.e., RJR-2403-sensitive component) current responses (summed as in the response to ACh) (B). C: typical traces of 10 mM choline-induced currents in MS/DB and VTA DAergic neurons showing different kinetics for current activation/desensitization with a slower response characteristic of MS/DB neurons. D: statistical comparisons of kinetics of 10 mM choline-induced currents in MS/DB cholinergic and VTA DAergic neurons. ***p<0.001.

Antagonist profiles for MS/DB and VTA nAChRs. Concentration-dependent block by MLA (at the indicated concentrations in nM after pre-exposure for 2 min and continued exposure during agonist application indicated by open bars) of 10 mM choline-induced (applied as indicated by closed bars) whole-cell currents (representative traces shown) in MS/DB (Aa) and VTA (Ab) neurons was not significantly different (p>0.05, Ac). However, choline-induced currents in MS/DB neurons (Ba) were more sensitive to block by DHβE (at the indicated concentrations in μM after pre-exposure for 2 min and continued exposure during agonist application indicated by open bars) than in VTA neurons (Bb; concentration-response profile shown in Bc).

Inhibition of choline-induced currents in dissociated MS/DB neurons by Aβ_1-42 was concentration-and form-dependent. A: Normalized, mean (± SE), peak current responses (ordinate) of the indicated numbers of MS/DN neurons as a function of time (abscissa, min) during challenges with choline in the presence of 1 nM scrambled Aβ(■) or in the presence of 0.1 nM (●), 1 nM (▲) or 10 nM (▼) Aβ show concentration dependence of functional block. B: Normalized responses (ordinate) during challenges with choline in the presence of 1 nM monomeric (■), oligomeric (▲) or fibrillar (●) Aβ indicate insensitivity to monomeric Aβ and highest sensitivity to peptide oligomers. *p<0.05, **p<0.01, and ***p<0.001.

Kinetics, pharmacology and Aβ sensitivity of α7-containing-nAChRs in nAChR β2 subunit knockout mice. Genotype analyses demonstrated that nAChR β2 subunits are not expressed in nAChR β2 knockout mice (A), whereas Lac-Z (as a marker for the knockout) was absent in wild-type (WT) mice (B). Kinetic analyses showed that whole-cell current kinetics and amplitudes differed for MS/DB neurons from WT compared to nAChR β2 subunit knockout homozygote mice (Ca,b). Compared to MS/DB neurons from WT mice (Da), choline-induced currents in MS/DB (Db) neurons from β2 knockouts were insensitive to DHβE but retained sensitivity to MLA (Dc). 1 nM Aβ_1-42 suppressed choline-induced currents in MS/DB neurons from WT (■) but not from β2 knockout (●) mice (E). ‘Control’ responses (▲) were choline-induced currents in neurons from WT mice without exposure to Aβ_1-42. *p<0.05, **p<0.01.

Identification of cholinergic neurons dissociated from basal forebrain. A: Phase contrast image of a rat MS/DB brain slice (region confirmed using The Rat Brain in Stereotaxic Coordinates, Paxinos and Watson, 1986). MS/DB neurons (phase-contrast images of dissociated neurons; B) exhibited spontaneous action potential firing (C), insensitivity to muscarine (C), action potential adaptation induced by depolarizing pulses (D), and did not show ‘sag’-like responses to hyperpolarizing pulses (E), suggesting they were cholinergic. F: Dissociated neuron (phase contrast, Ph) labeled with lucifer yellow (LY) showed positive ChAT immunostaining following patch-clamp recording.

nAChR α7 and β2 subunits are co-expressed, co-localize and co-assemble in rat forebrain MS/DB neurons. RT-PCR products from whole brain, VTA and MS/DB regions (A) corresponding to the indicated nAChR subunits or to the housekeeping gene GAPDH were resolved on an agarose gel calibrated by the flanking 100 bp ladders (heavy band is 500 bp) and visualized using ethidium staining. Note that the representative gel shown for whole brain did not contain a sample for the nAChR α3 subunit RT-PCR product, which typically is similar in intensity to the sample on the gel for the VTA and MS/DB. B: quantification of nAChR subunit mRNA levels for RT-PCR amplification followed by Southern hybridization with ³²P-labeled, nested oligonucleotides normalized to the GAPDH internal control and to levels of each specific mRNA in whole rat brain (ordinate: ± S.E.M.) for the indicated subunits. C: From 15 MS/DB neurons tested, after patch-clamp recordings (Ca: representative whole-cell current trace) the cell content was harvested and single-cell RT-PCR was performed, and the results show that α7 and β2 were the two major nAChR subunits naturally expressed in MS/DB cholinergic neurons (Cb-Cd). Double immunofluorescence labeling of a MS/DB neuron using anti-α7 and anti-β2 subunit antibodies revealed that α7 and β2 subunit proteins co-localized, and similar results were obtained using 31 neurons from 12 rats (D). Protein extracts from rat MS/DB (lane 1) or rat VTA (lane 2) or from MS/DB from nAChR β2 subunit knockout (lane 4) or wild-type mice (lane 5) were immunoprecipitated (IP) with a rabbit anti-α7 antibody (Santa Cruz H302; lanes 1, 2, 4, and 5) or rabbit IgG as a control (lane 3). The eluted proteins from the precipitates were analyzed by immunoblotting (IB) with rat monoclonal anti-β2 subunit antibody mAb270 (upper panel) or rabbit anti-α7 antisera H302 (lower panel). The β2 and α7 bands are indicated by arrows (E). All these data suggested that nAChR α7 and β2 nAChR subunits are co-assembled in MS/DB neurons.

Effects of 1 nM Aβ_1-42 on α7β2-nAChRs on MS/DB neurons. Typical whole-cell current traces for responses of MS/DB neurons to 10 mM choline challenge at the indicated times after initial challenge alone show no detectable rundown during repetitive application of agonist (2-s exposure at 2-min intervals; Aa). Choline-induced currents in rat MS/DB neurons were suppressed by 1 nM Aβ_1-42 (continuously applied for 10 min, but responses to challenges with choline are shown at the indicated times of Aβ exposure; Ab) but not by 1 nM scrambled Aβ_1-42 (as a control; Ac). Choline-induced currents in VTA neurons were not affected by 1 nM Aβ_1-42 (Ad). B: Normalized, mean (± SE), peak current responses (ordinate) as a function of time (abscissa, min) during challenges with choline alone (□), in the presence of 1 nM Aβ(▲), or in the presence of control, scrambled Aβ(▼) for the indicated numbers of MS/DB neurons, or during challenges with choline in the presence of 1 nM Aβ for the indicated number of VTA neurons (●) illustrate that only choline-induced currents in rat MS/DB neurons were sensitive to functional inhibition by Aβ.

Effects of Aβ on heterologously-expressed, homomeric α7-and heteromeric α7β2-nAChRs in Xenopus oocytes. Choline (10 mM, 2-s exposure at 2-min intervals)-induced whole-cell current responses in oocytes injected with rat α7-nAChR subunit cRNA alone (Aa, black trace) or with α7 and β2 subunit cRNAs at a ratio of 1:1 (Aa, gray trace) show slower decay of elicited currents and a longer decay time constant for heteromeric receptors (Aa and b). The scale bars represent 1 sec and 1 μA for the α7-nAChR response (black trace) and I sec and 100 nA for the α7β2-nAChR response (gray trace), thus also showing that current amplitudes were lower for heteromeric than for homomeric receptors. B: Normalized, mean (±SE), peak current responses (ordinate) of the indicated numbers of oocytes heterologously expressing nAChR α7 and β2 subunits (■, ●) or only α7 subunits (▲) as a function of time (abscissa, min) during challenges with choline alone (■) or in the presence of 10 nM Aβ (●, ▲) show sensitivity to functional block by Aβ only for heteromeric receptors. *p<0.05, **p<0.01, and ***p<0.001.