AFM offers structural information and real-time assessment of dynamic processes in living cells. However, its use to investigate neuronal interactions has been limited. Here, we used AFM to characterize the adhesion frequency and detachment forces of the neuronal acetylcholine receptor (nAchRs) on medullary respiratory neurons. Neurons from neonatal (P5-P10) and juvenile (3-4 wk) rats were enzymatically dispersed and cultured. Functionalized AFM probes were coated with an anti-α7 subunit antibody (0.5mg/ml) and used to repeatedly contact the cell surface (0.5 Hz, 100 contacts per cell). Nicotine (1 mM) added to the culture media significantly reduced AFM binding probability (31.3 ± 7.8% vs. 5.9 ± 2.1%, P<0.05, n=9). Similarly, α-bungarotoxin (1 mM), an α-7 subunit containing nAchR inhibitor, significantly reduced binding probability (13.0 ± 3.3 vs 4.1 ± 0.7%, P<0.05, n=10). Peak force measurements were similar between the first 5 and last 5 contacts (47.3 ± 1.1 vs. 45.6 ± 1.4 pN, p=0.404, n=10) demonstrating that probe binding did not decay over time. Internalization of Alexa Fluora 488-substance P (AF488-SP) was used to identify neurokinin-1 receptors as a marker of respiratory neurons. AF488-SP was visualized in all neurons tested. These results demonstrate that AFM can be successfully used to detect nAChR on the surface membrane of living medullary neurons. [ABSTRACT FROM AUTHOR]