A single application of nicotine induces sustained changes in [Ca²⁺]_i along vHipp axons.

<div><p>Spinning disk confocal live Ca²⁺ images from WT vHipp axons were recorded every 10 seconds for 30 min and fluo-4 fluorescence intensities were calculated and quantified as a normalized integrated intensity at each time point. The changes of normalized integrated intensities at one spot of vHipp axons were plotted vs. time. </p> <p><b>A1</b>: Representative plot from a live WT vHipp axon perfused with normal HEPES buffered solution showed that the normal axonal intracellular Ca²⁺ signals oscillated in a random manner with very small amplitude. <b>A2</b>: Representative spinning disk confocal fluo-4 images are indicated on pseudo color scale at different time points (0, 2.1’, 4.5’, 17.5’, 20.5’, and 23’). The square area is the region for which fluo-4 intensities were quantified. Scale bar: 5μm. <b>A3</b>: Representative spinning disk confocal fluo-4 images are indicated on pseudo color scale in 2D manner from the square area (see A2) at different time points.</p> <p><b>B1</b>: Representative plot from a live WT vHipp axon perfused with nicotine (1μM) for 1 min and then with normal HEPES buffered solution without nicotine. The nicotine application induced both a quick and sustained increase of axonal intracellular calcium signals that oscillated in a random manner with large amplitude. <b>B2</b>: Representative spinning disk confocal fluo-4 imagings are indicated on pseudo color scale at different time points after nicotine application (0, 2’, 4’, 17.5’, 22’, and 25’). The square area is the area for which fluo-4 intensities were quantified. Scale bar: 5 μm. <b>B3</b>: Representative spinning disk confocal fluo-4 images are indicated on pseudo color scale in 2D manner from the square area (see B2) at different time point.</p> <p><b>C</b>: After recording of Fluo-4/Ca²⁺fluorescence (<b>c1</b>), the culture of vHipp microslices were fixed, permeabilized, and stained with antibodies recognizing panaxonal marker (green), MAP2 (red),and GFAP (blue). The vHipp projections previously assessed by calcium imaging and relocated post hoc are co-labeled by panaxonal marker (<b>c2</b>), but not by either a dendritic marker MAP2 (<b>c3</b>), or by a glia marker GFAP (<b>c4</b>), scale bar: 5μm. </p></div

Activation of PLC and CaMKII is required for the sustained, nicotine induced change in intracellular Ca²⁺ in vHipp axons.

<p>Box plot of pooled data on nicotine induced Ca²⁺ signaling [Δ<i>F</i>/ F_<i>0</i>= (F - F_<i>0</i>)/F_<i>0</i>] after 30 minutes of pre-incubation with inhibitors of CaMKII, (KN93; 5 μM, 8 recordings in 8 coverslips from 6 mice andAutocamtide-2-Related Inhibitory Peptide; AIP, 20 μM, 6 recordings in 6 coverslips from 4 mice). Although CaMKII inhibitors had no effect on the acute phase of Ca²⁺ signaling, the sustained phase was completely blocked by pre-incubation with KN93 or AIP. A 30 min pre-incubation with an inhibitor of phospholipase C (U73122, 10 μM, 8 recordings in 6 coverslips from 4 mice) or Src tyrosine kinase inhibitor (PP2, 20 μM, 6 recordings in 6 coverslips from 4 mice) did not affect the acute phase of nicotine induced Ca²⁺ signaling. In contrast, the sustained phase was blocked by U73122, but not by PP2. At least 1500 μm axonal lengths for each group were collected and quantified. **p<0.01 .</p

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. </p

Model showing role of presynaptic α7*nAChR mediated calcium signaling in sustained facilitation of synaptic transmission.

<p><b>Step 1</b>, Activation of presynaptic nAChRs by nicotine induces calcium influx into the presynaptic terminals. Activation of non-α7*nAChR (low Ca²⁺-permeability) elicits small, acute increases in intracellular Ca²⁺ at presynaptic terminals. <b>Step 2</b>, Activation of α7*nAChR (high Ca²⁺-permeability) elicits a larger increase in intracellular Ca²⁺ at presynaptic terminals which is sufficient to induce calcium release from the IP₃-sensitive internal Ca²⁺ stores and maintain sustained changes in intracellular Ca²⁺; activation of α7*nAChR also can activate PLC by unknown mechanisms. PLC generates IP₃ which mobilizes IP₃ receptor controlled calcium stores (<b>Step 3</b>)<b>. Step4</b>, CaMKII is also activated by activation of α7*nAChR, and active CaMKII in turn, via a positive feedback mechanism, sustains the elevation of presynaptic Ca²⁺. <b>Step 5 and 6</b>, The sustained change in intracellular Ca²⁺ and activation of CaMKII at presynaptic terminals triggers the long-term enhancement of glutamate release, inducing long-term facilitation of glutamatergic synaptic transmission through glutamate receptors.</p

CICR is required for the nicotine induced sustained response in intracellular Ca²⁺ in vHipp axons.

<p>Box plot of pooled data shows that the acute effects of nicotine [Δ<i>F</i>/ F_<i>0</i>= (F - F_<i>0</i>)/F_<i>0</i>] were not altered by 30 min pre-incubation with a blocker of ryanodine-sensitive endoplasmic reticulum stores, (ryanodine 30 μM, 9 recordings in 9 coverslips from 7 mice) or an inhibitor of IP₃ receptors, (xestospongin-c 100 nM, 8 recordings in 8 coverslips from 6 mice). In contrast, the sustained change in intracellular Ca²⁺ seen at 30 min after nicotine treatment at WT vHipp axons was still seen in ryanodine treated cultures but not in xestospongin-c treated cultures. At least 1500 μm axonal lengths for each group were collected and quantified. **<i>p</i>< 0.01.</p

α7*nAChRs participate in nicotine induced sustained changes in intracellular Ca²⁺ in vHipp axons.

<div><p><b>A</b>: Schematic of experimental protocols. a1: Spinning disk confocal images from live WT and/or α7-/- vHipp axons were recorded every 10 seconds for 30 min, including baseline data collection for 5 minutes, and followed by application and washout of nicotine or α7*nAChR or non-α7*nAChR agonist. a2: To dissect out the subtypes of nAChR involved in sustained changes in intracellular Ca²⁺ elicited by nicotine, WT vHipp axons were pre-incubated with an α7*nAChR antagonist (αBgTx) or with a non-α7*nAChR antagonist (DHβE) for 25 minutes respectively, after which the protocol illustrated in part <b>a1</b> was followed. a3: WT axonal calcium signals were recorded at baseline followed by nicotine application and wash out. Ten minutes after nicotine application, the α7*nAChR antagonist (αBgTx) was added to address whether inhibition of α7*nAChRs can block Ca²⁺ signaling once the process was initiated.</p> <p><b>B</b>: Representative spinning disk confocal fluo-4 images in pseudo color scale before (Top), 1’ (Middle), and 30’ (Bottom) after nicotine application to WT (<b>left</b>) and α7-/- (<b>right</b>) vHipp axons. Scale bar: 5μm. </p> <p><b>C</b>: Box plot of pooled data shows that the acute effects of nicotine on fluo-4 fluorescence (<b><i>ΔF/F₀= (F-F₀)/F₀</i></b>) were comparable for WT (27 recordings in 21 coverslips from 15 mice) and α7-/- (15 recordings in 13 coverslips from 10 mice) vHipp axons. In contrast, the sustained change in intracellular Ca²⁺ seen at 30 min after nicotine treatment of WT vHipp axons was not seen in α7-/- vHipp axons. Preincubation with αBgTx (11 recordings in 10 coverslips from 9 mice) eliminated the sustained phase of nicotine induced intracellular Ca²⁺ response, whereas the DHβE (11 recordings in 10coverslips from 8 mice) did not. RJR-2403 (7 recordings in 7 coverslips from 7 mice) elicited only the acute phase of Ca²⁺ signaling whereas PNU282987 (7 recordings in 7 coverslips from 7 mice) caused sustained Ca²⁺ signaling. Application of αBgTx 10 minutes after nicotine washout had no effect on the subsequent Ca²⁺ response. At least 1500 μm axonal lengths for each group were collected and quantified. **<i>p</i><0.01. </p> <p><b>D</b>: α7*nAChR clusters are co-localized with the “hot spots” of nicotine-induced sustained Ca²⁺ response along vHipp axons. After recording of nicotine-induced changes of Fluo-4/Ca²⁺ fluorescence (<b>d1, d2, d3</b>), the vHipp axons were labeled for surface α7*nAChR with αBgTx–Alexa 594 (<b>d4</b>). Relocalization of sites at which nicotine had induced sustained changes in Ca²⁺ signaling along vHipp axons (<b>white arrow in d3</b>) revealed that these sites corresponded to sites of positive staining for surface α7*nAChR (<b>white arrow in d4</b>). Relocalization of sites where nicotine elicited only acute changes in Ca²⁺ signaling (<b>orange arrow in d2</b>) were not labeled by αBgTx–Alexa 594 (<b>d4</b>). Scale bar: 10μm. </p></div

Dissociated sensory neurons from E11 chick embryos were treated with B2-ECD (control) or B4-ECD for 1 h

In parallel, neurons were pretreated with WM or an Akt inh. for 45 min before treatment with B2-ECD or B4-ECD for an additional hour. Neurons were labeled for surface α7* nAChRs with αBgTx-488 (green), fixed, permeabilized, and costained for NF protein (blue). (A) Representative micrographs of αBgTx-488 staining along NF-positive axons. B4-ECD treatment increased surface αBgTx-488 clusters (b), which did not occur in the presence of WM (d). Linescans of fluorescence intensity profiles of αBgTx-488 along representative axons (see Materials and methods) are shown. Bar, 5 μm. (B) Quantification of surface αBgTx-488 clusters along sensory neuron axons represented in A. B4-ECD treatment induced an ∼1.9-fold increase of surface αBgTx-488 clusters but not in the presence of WM or Akt inh. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.0001 (Statview). (C) Quantification of surface αBgTx-488 cluster area. B4-ECD treatment induced an increase in αBgTx-488 cluster area but not in the presence of WM or Akt inh. Data pooled from three independent experiments were analyzed using nonparametric statistics and presented as box plots (see Materials and methods). Statistical significance determined by the Kolmogorov-Smirnov Test. *, P = 0.0001 (Statview).<p><b>Copyright information:</b></p><p>Taken from "Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons"</p><p></p><p>The Journal of Cell Biology 2008;181(3):511-521.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364689.</p><p></p

Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons-1

Re labeled for surface α7* nAChRs with αBgTx-488 (green), fixed, permeabilized, and labeled for NF protein (blue). (A) Representative micrographs of axons from WT (a and b) or Type III Nrg1 (c and d) sensory neurons under control (a and c) versus B4-ECD (b and d) conditions. B4-ECD treatment increased the number of surface αBgTx-488 clusters along NF-positive processes of WT neurons (b). Linescans of fluorescence intensity profile for αBgTx-488 staining along representative axons (see Materials and methods). Bar, 5 μm. (B) Quantification of surface αBgTx-488 clusters along NF-positive axons from WT versus Type III Nrg1 DRG explants treated with either B2-ECD (control) or B4-ECD for 24 h. In WT cultures, B4-ECD treatment induced an ∼1.6-fold increase in surface αBgTx clusters along NF-positive axons compared with the control. There was no detectable change in αBgTx clusters along axons of Type III Nrg1 neurons. The graph shows means ± SEM. Data were pooled from three independent experiments. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.03; **, P < 0.001 (Statview). (C) After 2 d in vitro, dissociated sensory neurons from E11 chick embryos were treated with B2-ECD (control) or B4-ECD for 1, 2, 6, or 12 h and labeled as described in A. Axonal surface αBgTx-488 clusters were quantified. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Statview). (D) Axonal-bound B4-ECD and αBgTx-488 were detected in puncta along axons treated with B4-ECD (b, d, and e). Sensory neurons from E14.5 WT mouse embryos were cultured for 2 d in vitro and treated with B2-ECD (control) or B4-ECD for 1 h. Before fixation, surface α7* nAChRs and axonal-bound B2-ECD (control) or B4-ECD were labeled with αBgTx-488 (green) and an antibody against the human Fc domain (anti-Fc; red), respectively. c and d and e are magnifications of the areas shown in dotted squares in a and b, respectively. Bar: (a and b) 5 μm; (c–e) 1 μm. (E) Sensory neurons from E11 chick embryos were treated with B2-ECD (control), B4-ECD, or soluble Nrg1β peptide (Nrg1-ECD) for 1 h. In parallel, neurons pretreated with an ErbB tyrosine kinase inhibitor (ErbB inh.) for 45 min were treated with B2-ECD, B4-ECD, or Nrg1-ECD for 1 h. Neurons were labeled as described in A, and surface αBgTx-488 clusters along axons were quantified. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.005; **, P < 0.01 (Statview).<p><b>Copyright information:</b></p><p>Taken from "Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons"</p><p></p><p>The Journal of Cell Biology 2008;181(3):511-521.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364689.</p><p></p

(A) Dissociated sensory neurons from E11 chick embryos were treated for 5 min with B2-ECD (control), B4-ECD, 50 ng/ml NGF, or 10 ng/ml of soluble Nrg1β peptide (Nrg1-ECD)

In parallel, neurons were treated with WM for 45 min before B4-ECD stimulation (WM + B4-ECD). Neurons were fixed, permeabilized, and costained for PIP (red) and tau protein (blue) to label axons. Both B4-ECD (g) and NGF (i) treatment induced puncta of PIP along tau-positive axons. Neither B4-ECD stimulation in the presence of WM (c and h) nor that of Nrg1-ECD (e and j) induced an increase in PIP. Confocal images were obtained with a 40× oil objective. Bar, 10 μm. (B) Immunoblot analysis of phospho-Akt (Ser 473) in WT or Type III Nrg1 sensory neurons treated with either B2-ECD (control) or B4-ECD for 10 min. In WT neurons, B4-ECD treatment induced an approximately threefold increase in phospho-Akt, whereas no response was detected in mutant neurons. Total Akt in the bottom panel shows equal lysate loading. The bar graph represents phospho-Akt normalized to total Akt immunoreactive bands. Data are representative of three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.002 (Statview). (C and D) E14.5 WT (a and b) or Type III Nrg1 (c and d) DRG explants were treated with B2-ECD (control) or B4-ECD for 10 min. Surface-bound B4-ECD or B2-ECD were labeled with an antibody against the human Fc domain (anti-Fc; green) before fixation. Neurons were fixed, permeabilized, and stained for phospho-Akt (red) and NF protein (blue). B4-ECD treatment increased phospho-Akt along Fc-positive axons of WT neurons (b and D) but did not do so along axons of mutant neurons (d). Note the close proximity of anti-FC and phospho-Akt puncta in the high-power micrograph shown in e. The asterisk denotes an axon negative for both anti-Fc and phospho-Akt immunolabeling (c). A 63× oil objective was used (a–d). Confocal imaging was obtained with a 100× oil objective (D). Bar: (a–d)10 μm; (D) 5 μm. (E) Quantification of the average fluorescence intensity (AFI) of phospho-Akt along axons of WT or Type III Nrg1 sensory neurons treated with B2-ECD (control) or B4-ECD for 10 min or 1, 2, or 6 h (see Materials and methods). Along WT axons, B4-ECD treatment induced increases in phospho-Akt. Along axons of mutant neurons, we did not detect an increase in phospho-Akt in response to B4-ECD treatment. The graph shows means ± SEM. Data are from three independent experiments. Statistical significance was determined by ANOVA. *, P < 0.02.<p><b>Copyright information:</b></p><p>Taken from "Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons"</p><p></p><p>The Journal of Cell Biology 2008;181(3):511-521.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364689.</p><p></p

Dissociated sensory neurons from E11 chick embryos were cultured for 2 d in vitro and treated with either B2-ECD (control) or B4-ECD for 1 or 24 h

(A) Quantification of surface or total pools of α7* nAChR by I-αBgTx radiolabeling in sensory neurons treated with either B2-ECD (control) or B4-ECD for 24 h. In response to a 24-h B4-ECD treatment, we detected an ∼2.7-fold increase in surface I-αBgTx binding compared with control conditions (B2-ECD [control], 1,339.15 ± 329.77 cpm; and B4-ECD, 3,562.81 ± 1,111.19 cpm). B4-ECD treatment did not induce a change in total I-αBgTx binding as compared with the control (B2-ECD [control], 11,159.74 ± 1,059.79 cpm; and B4-ECD, 12,258.85 ± 580.11 cpm). The graph shows means ± SEM. Data were pooled from three independent experiments with greater than or equal to three wells per condition per experiment. Statistical significance was determined by ANOVA. *, P < 0.05 (Statview). (B) Immunoblot analysis of total α7 subunit protein in sensory neurons treated with B2-ECD (control) or B4-ECD treatment for 24 h. In response to B4-ECD treatment, we did not detect a difference in total α7 subunit protein. NF probing in bottom panel shows equivalent lysate loading. (C) Sensory neurons were treated with B2-ECD (control) or B4-ECD for 1 h. In parallel, neurons pretreated with CHX for 45 min were treated with B2-ECD or B4-ECD for 1 h. Neurons were labeled with αBgTx-488 (green), fixed, permeabilized, and colabeled for NF protein (blue). CHX treatment (c and d) did not affect either the basal number of αBgTx-488 clusters on control neurons (c) or the response to B4-ECD (d). Linescans of fluorescence intensity profiles of αBgTx-488 along representative axons (see Materials and methods) are shown. Bar, 5 μm. (D) Quantification of surface αBgTx-488 clusters along NF-labeled axons. B4-ECD treatment induced an ∼1.9-fold increase in surface αBgTx-488 clusters along axons, and B4-ECD treatment in the presence of CHX induced an ∼2.1-fold increase. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P = 0.01; **, P < 0.0001 (Statview). (E) Quantification of surface αBgTx-488 cluster area. B4-ECD treatment in the presence or absence of CHX induced an increase in αBgTx-488 cluster area. Data pooled from three independent experiments were analyzed using nonparametric statistics and presented as box plots (see Materials and methods). Statistical significance was determined by the Kolmogorov-Smirnov Test. *, P ≤ 0.0001 (Statview).<p><b>Copyright information:</b></p><p>Taken from "Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons"</p><p></p><p>The Journal of Cell Biology 2008;181(3):511-521.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364689.</p><p></p