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) and a proprietary software package (NforceR). Force curves where this website no rupture event was detected were counted as non-adhesion events. Single bond strengths were adjusted using probe sensitivity and spring constants as covariates. As no differences were found in binding probability between cultures obtained from neonatal or juvenile rats (0.25 �� 0.07 versus 0.13 �� 0.05, neonatal versus juvenile, P= 0.12), data were combined and analysed. The difference between means for the effect of a given treatment was determined using Student's paired t test or ANOVA. All statistical analyses were performed using PC SAS for Windows (version 9.0; SAS Institute, Cary, NC, USA). Differences were considered significant at a level of P Nutlin-3 cell line (?11.90 to ?12.88 mm from bregma). Figure 1 illustrates the location of a tissue punch from one brain section counterstained using Neutral Red. Note that the punch was located immediately ventral and lateral to the nucleus ambiguus in a region that contained high levels of NK1-R immunofluorescence. The presence of high NK-1R immunoreactivity in this region of the ventrolateral medulla has been suggested to be a marker for central respiratory neurons, including pre-B?tC neurons (Gray et al. 1999, 2001; Wang et al. 2001; Fong & Potts, 2006). The AFM experiments were performed only on neurons that exhibited NK1-R fluorescence (Fig. 1C). To test nAchR function, the AFM probe was coated with anti-��7 nAChR antibody. Using this approach, computer-controlled probing of the surface of the soma via AFM was performed to obtain a series of force curves (Fig. 2; see Methods section). To assess the specificity of AFM probe binding, a specific antagonist of ��7 subunit-containing nAChRs, INPP5D ��-bungarotoxin, was added to the culture media. Bath application of ��-bungarotoxin significantly reduced binding probability (13.0 �� 3.3 versus 4.1 �� 0.7%, control versus��-bungarotoxin, P