α7*nAChR and CICR are required for sustained, nicotine induced activation of CaMKII along vHipp axons.

<p>vHipp microslice cultures from WT mice were fixed after incubation with nicotine, and then permeabilized, and stained with antibodies recognizing phospho-CaMKII (red) and axonal neurofilaments (green). <b>A</b>: Representative micrographs of WT vHipp axons are shown above line scans of fluorescence intensity profile for phospho-CaMKII staining; control <i>vs</i>. 5 min after nicotine (Scale bar: 10 μm). <b>B</b>: phospho-CaMKII immunofluorescent intensities along axons were quantified (as ratio of phospho-CaMKII/pan-axonal marker per 100 μm axons) under control (0.86±0.06) or after nicotine application (1.65± 0.08, **<i>P</i>< 0.01, One-Way ANOVA Post Hoc Tests). KN93, a CaMKII inhibitor, had no effect on basal phospho-CaMKII immunofluorescent intensities (0.76± 0.02), but blocked nicotine activation of CaMKII (0.80± 0.04). Total CaMKII immunofluorescent intensities along axons were also quantified (as ratio of tCaMKII/pan-axonal marker per 100 μm axons) under control (1.08±0.19) or after nicotine application (1.12± 0.24, <i>P</i>> 0.05, One-Way ANOVA Post Hoc Tests). <b>C</b>: The time-course of CaMKII activation was quantified. vHipp axons were fixed in 1, 5, 15, 30, and 60 min respectively after nicotine treatment. phospho-CaMKII immunofluorescent intensities along vHipp axons increased quickly (1.65± 0.08 at 1’) and remained elevated for at least 30 min (1.75± 0.12 at 5’, 1.76± 0.09 at 15’, and 2.00± 0.10 at 30’, **<i>P</i>< 0.01, One-Way ANOVA Post Hoc Tests), returning to control levels at 60 min (0.84± 0.04). <b>D</b>: vHipp microslices culture from either WT or α7-/- mice were fixed 5 min after the incubation of nicotine (with 15’ pre-incubation of αBgTx or DHβE) or α7*nAChR selective agonist choline (10 mM for 1 min). αBgTx (100 nM), α7*nAChR selective antagonist, blocked (1.11± 0.08) the nicotine activation of CaMKII in vHipp axons, but DHβE (1 μM), the non-α7*nAChR selective antagonist, did not (1.60± 0.12). Choline (10 mM), the selective α7*nAChR agonist, activated (1.76± 0.04) axonal CaMKII. Neither nicotine (1μM, 0.88± 0.04) nor choline (10 mM, 0.86± 0.08) activated CaMKII in vHipp axons from α7-/- mice. <b>E</b>: vHipp microslice cultures from WT mice were fixed 5 min after nicotine treatment with 30 min pre-incubation of CdCl₂, ryanodine or Xes-C. Pre-incubation with CdCl₂ (100 μM, 1.61± 0.08), a voltage-gated calcium channel blocker, or ryanodine (30 μM, 1.62± 0.11), an intracellular calcium store ryanodine receptor blocker, did not affect nicotine activation of CaMKII in vHipp axons. Pre-incubation with Xes-C (100 nM, 0.90± 0.04), an intracellular calcium store IP₃ receptor blocker, prevented nicotine activation of CaMKII. Data represent the mean ± SEM, **<i>P</i>< 0.01, One-Way ANOVA Post Hoc Tests. At least 1500 μm axonal lengths for each group from three independent experiments were collected and quantified.