Nicotinic Acetylcholine Receptors in Mouse and Rat Optic Nerves

Chuan-Li Zhang, Yakov Verbny, Sameh A. Malek, Peter K. Stys, Shing Yan Chiu


Receptor-mediated calcium signaling in axons of mouse and rat optic nerves was examined by selectively staining the axonal population with a calcium indicator. Nicotine (1-50 μM) induced an axonal calcium elevation that was eliminated when calcium was removed from the bath, suggesting that nicotine induces calcium influx into axons. The nicotine response was blocked by d-tubocurarine and mecamylamine but not α-bungarotoxin, indicating the presence of calcium permeable, non-α7 nicotinic acetylcholine receptor (nAChR) subtype. Agonist efficacy order for eliciting the axonal nAChR calcium response was cytisine ∼ nicotine >> acetylcholine. The nicotine-mediated calcium response was attenuated during the process of normal myelination, decreasing by approximately 10-fold from P1 (premyelinated) to P30 (myelinated). Nicotine also caused a rapid reduction in the compound action potential in neonatal optic nerves, consistent with a shunting of the membrane after opening of the nonspecific cationic nicotinic channels. Voltagegated calcium channels contributed little to the axonal calcium elevation during nAChR activation. During repetitive stimulations, the compound action potential in neonatal mouse optic nerves underwent a gradual reduction in amplitude that could be partially prevented by d-tubocurarine, suggesting an activity-dependent release of acetylcholine that activates axonal AChRs. We conclude that mammalian optic nerve axons express nAChRs and suggest that these receptors are activated in an activity-dependent fashion during optic nerve development to modulate axon excitability and biology.


Recent studies have demonstrated that mammalian axons express at least three molecular pathways for mediating calcium influx: voltage-gated calcium channels (Callewaert et al. 1996; Fern et al. 1995; Jackson et al. 2001; Lev-Ram and Grinvald 1987; Sun and Chiu 1999), reverse operation of Na/Ca exchangers under ischemic conditions (Stys et al. 1995), and the calcium permeable vanilloid receptor TRPV1 (Fischer et al. 2003). Axonal calcium influx can trigger diverse cellular processes, including stimulating axonal transport, modulation of excitability, long-term changes in intracellular biochemistry, and excitotoxicity under pathological conditions (Breuer et al. 1992; Callewaert et al. 1996; Stys et al. 1995; Sun and Chiu 1999; Verbny et al. 2002). In this study, we describe a third pathway for mediating calcium influx into mammalian axons of the optic nerves: ligand-gated neurotransmitter receptors. Two mechanisms by which neurotransmitter receptor activation mediates calcium influx are considered. The first mechanism is that axonal receptor activation causes a membrane depolarization, which indirectly promotes calcium influx through activation of voltage-gated calcium channels. Indirect calcium entry is possible in mammalian optic nerve axons since they are known to express GABAA receptors (Sakatani et al. 1992), whose activation will depolarize axonal membrane and activate calcium channels. The focus of this paper is on the second mode of calcium entry occurring directly via calcium permeable axonal neurotransmitter receptors.

The possibility that axons express receptors that directly mediate calcium influx was first suggested in earlier studies (Armett and Ritchie 1961) that demonstrated that acetylcholine exerts a depolarizing action on nonmyelinated vagus nerves in rabbits. Subsequently, it has been established that one subtype of acetylcholine receptor, the nicotinic acetylcholine receptor (nAChR), is a ligand-gated, calcium permeable ion channel well documented to be present in both pre- and postsynaptic membranes. A recent calcium imaging study (Edwards and Cline 1999) directly investigated the possibility for nAChR-mediated calcium influx in nonsynaptic regions by focusing on nicotine-induced calcium signaling in axon arbors of the developing frog optic tectum. Intriguingly, Edwards and Cline (1999) observed nAChR-mediated calcium influx in nonsynaptic regions that correlates with sites of axonal branching. Since local rise in calcium may trigger actin/cytoskeletal assembly (Bentley and O'Connor 1994; Lankford et al. 1996), Edwards and Cline (1999) hypothesized that nAChR-mediated calcium influx regulates branching in growing retinal axons. Histological evidence previously demonstrated that AChRs containing beta 2 subunits are present in the retinal ganglion cells and transported along the optic nerve, presumably destined for insertion in the nerve terminals (Swanson et al. 1987). However, an important question is whether nAChR is actually inserted along the entire axon and plays a role in axon biology.

We set out to address this issue by using mouse and rat optic nerves excised between the eyes and optic chiasm, which are completely devoid of synaptic elements, branching, and growth cones. Using a method developed in our laboratory for selectively loading calcium indicators into the axons (Verbny et al. 2002), we examined if nAChR-mediated calcium signaling can be detected in these axons, and if so, how it relates to axonal excitability. Our results suggest that nAChRs are present in optic nerve axons. Furthermore, we characterized the antagonist profile of nAChRs, examined the relationship between nAChR expression and myelination, and provided evidence for activity-dependent release of acetylcholine in the neonatal mammalian optic nerves.


Selective axonal staining

In this paper, we used an in vitro method developed by Verbny et al. (2002) to selectively stain axons with calcium indicators. This in vitro method involved locally applying a high concentration of the cell-impermeant form of Oregon Green BAPTA-1 to the cut end of an excised optic nerve through a tight suction pipette and allowed approximately 3 h for the dyes to load into the axonal cylinders. We used Oregon Green BAPTA-1 conjugated to the high molecular weight (MW) dextran (MW =10,000) to exclude movement of dyes through glial gap junctions and to allow better retention in axons (Verbny et al. 2002).

Most of the experiments were performed on mice (about 120 mice), with a small number of experiments performed on rats (6 rats) when the length of the optic nerve became an issue. Each animal was killed, and optic nerves were taken out for study. In the rat optic nerve, the mean diameter of the axons remains about 0.2 μm for the first postnatal week and increases rapidly to about 1 μm in adults (>P28 days after birth), when the axons are fully myelinated (Foster et al. 1982). No morphometric data exist for the mouse optic nerves, and the developmental profile for the axon diameters is assumed to be similar to rats. Briefly, optic nerves excised between the eye and the chiasm were obtained from P1-P30 mice and laid down on the bottom of a perfusion chamber. The distal end of the nerve trunk was loosely drawn into a stimulating pipette, and the proximal end was drawn tightly into a recording pipette. The nerves were allowed to stabilize for 60 min before dye loading began. For dye loading, the normal saline solution in the recording pipette (the one with the cut end of the nerve tightly drawn in) was replaced either with a high-K (140 mM) or a high-Na (140 mM), low-calcium (0 calcium with or without 1 mM EGTA) solution containing 4-5 μl of the cell impermeant form of calcium indicators. The axons were stained by diffusion of the dyes from the cut end into the axonal cylinders. In our earlier studies, we used 1 mM EGTA in the loading pipette because we reasoned that this may prevent the cut ends of the axons from resealing, thereby facilitate dye loading into the axon cylinders. In latter studies, we found that robust axonal staining was also achieved without EGTA, and we routinely omitted EGTA in the loading pipette. The nicotine-induced calcium responses were not affected by the presence or absence of EGTA in the loading pipette. A standard loading time of 3 h was allowed before experiments began. We typically performed calcium imaging at a site approximately 500-1,000 μm away from the dyeloading pipette. The dyes were allowed to remain in the loading pipette (also serve as the recording pipette) during the entire experiment. Calcium levels were not reported in absolute levels, but as ΔF/F0, where F0 is the baseline fluorescence signal before drug application.

Confocal fluorescence imaging of axonal calcium

Calcium images were viewed with a 40× (Olympus) objective lens on a Noran Odyssey confocal system (Madison, WI). For calcium indicators, we used the cell-impermeant, high-affinity Oregon Green 488 BAPTA-1 (Kd = 170 nM; MW = 1,114) conjugated to the high MW dextran (MW = 10,000; Molecular Probes). The reason for using the dextran-conjugated dye is that dextran cannot pass through gap junctions (Eckert et al. 1999), thus excluding contribution from glial signals resulting from transfer of dyes from glial cells at the site of loading to the site of optical recording via gap junctions (Verbny et al. 2002). As argued in Verbny et al. (2002), the absence of an adenosine-evoked calcium response, a hallmark for glial cells, demonstrates that selective staining of axons has been achieved with this method. All dyes were used at a concentration of 5 mM in the loading pipette; the actual axonal dye concentration at optical recording site was estimated in our previous study (Verbny et al. 2002) to be approximately 1.3% of the dye concentration at the loading pipette, which would be about 65 μM. Experiments were performed with the Noran Odyssey System, with the dyes excited by an argon laser at 488 nm and confocal fluorescence signals collected through a 500-nm long-pass emission filter. In most studies, the average fluorescence signal from the whole field (approximately 10,000 μm2 area) was collected on-line at near video rate (30 Hz) and stored for off-line analysis. In other studies, images were captured of individual or groups of axons for image analysis of calcium changes. Image acquisition and on-line calculations were controlled through the Metamorph software (Universal Imaging). Experiments were done at room temperature (20-22°C).


Compound action potentials (CAPs) were evoked by a 125% supramaximal stimulus applied via the suction electrode to the cut end and recorded from a second suction electrode at the other cut end. The amplitude of the CAP data was analyzed using Pclamp 6.0 software (Axon Instruments). Compound axonal resting potentials were measured in neonatal rat optic nerve using the grease gap technique as previously described (Stys et al. 1995). Briefly, optic nerves were excised and inserted into a silastic tube filled with petroleum jelly, spanning two compartments. One was perfused with Ringer solution and test compounds, and the other was perfused with isotonic K solution where NaCl was replaced with KCl. A stable steady-state potential develops across the two wells that is a reliable fraction of the true axonal resting potential of the inserted fiber bundle. Typical absolute potentials recorded from P7 rat nerves were in the range of -25 to -35 mV.

Solution and drugs

The optic nerves were normally bathed in a Ringer solution containing (in mM) 129 NaCl, 3 KCl, 1.2 NaH2PO4, 2.4 CaCl2, 1.3 MgSO4, 3 HEPES, 20 NaHCO3, and 10 glucose. All solutions were rigorously oxygenated with 95% O2-5% CO2. Calcium-free solutions were prepared by replacing Ca2+ with Mg2+ to keep the external divalent cation concentration constant and by adding EGTA (1 mM); pH was adjusted to 7.4 with NaOH or HCl as necessary.

All compounds were from Sigma. The handling of the chemicals and animals was approved by the Biological Safety and Animal Research Committee at the University of Wisconsin, Madison.


Selective staining of axons

Representative axonal stains in this study are shown in Fig. 1 for neonatal (Fig. 1B, left) and adult (Fig, 1, C and D, right) optic nerves. In most cases, a large number of axons were stained. Most stained axons do not stay in the same focal plane, making it difficult to follow a single axon for its entire length along the optic nerve. Figure 1D shows example of an occasional stained axon that resided in the same focal plane for ≤200 μm within the field of view. We also verified axonal staining by an in vivo method in which we injected Oregon-Green-Dextran into the vitreous space of the eye of an adult mouse in vivo and allowed 24 h for the dyes to be taken up by the ganglion cells and transported down the axons, killed the animal, and mounted the optic nerve for image analysis. Single axons stained by this in vivo injection method (Fig. 1E) have morphology that is identical to that stained by the in vitro suction pipette method. In general, the staining method using suction pipette produces large populations of dye-loaded axons for analysis compared with the in vivo injection that only produces occasional stained axons. The pattern of axonal staining can be compared with the staining obtained by injecting the cell-permeant form of the dye into the nerve trunk (Fig. 1A), which also labeled glial cells (Kriegler and Chiu 1993). Since we are primarily interested in the axonal response to nicotine in this work, our analysis is based only the axonal staining pattern exemplified by Fig. 1, B-D. Since the in vitro loading method was developed for the mouse optic nerves, most of the experiments were performed in the mouse optic nerves. However, where necessary when nerve size becomes a technical issue, the larger rat optic nerves were used.

fig. 1.

Staining of axons in mouse optic nerves with calcium indicators. A: images of glial cells stained with the method of Kriegler and Chiu (1993). Age P4. Glial cells were stained by injecting the cell permeant form of Oregon-Green-BAPTA into the nerve trunk. Bar is 10 μm. B-D: images of dye-loaded axons obtained using the loading method of Verbny et al. (2002). Animal age: P4 (B) and P60 (C and D). D: occasional presence of a single stained axon that can be followed through the entire field of view of the focal plane. Axons were loaded by locally applying the cell impermeant Oregon-Green-BAPTA-dextran through the cut ends of the nerve via a tight suction pipette for 3 h. Bar is 10 μm in B and 20 μm in C and D. E: image of a single dye-loaded axon stained by in vivo injection of Oregon-Green-dextran into the vitreous space of the eye of an adult mouse. The animal was killed 24 h after injection, and the optic nerve mounted for calcium imaging. Bar is 20 μm.

nAChR-mediated axonal calcium influx in mouse optic nerves

Figure 2 shows pseudo-color image of a single axon from a P8 optic nerve before (Fig. 2A) and about 40 s after (Fig. 2B) bath application of 50 μM nicotine. Figure 2, C and D, shows the same images recomputed as ΔF/F0 on a pixel-by-pixel basis, a procedure that normalizes for differences in the resting signal due to differences in dye loading and to changes in focus as the axon slightly goes in and out of the focal plane. Nicotine induces calcium elevation throughout the axon (Fig. 2, B and D). The goal of this paper is to characterize this nicotine response. In all the subsequent experiments, we captured the population fluorescence from the entire field of view without discerning individual axons to achieve a much faster time resolution (population signal sampled every 30-50 ms) than image analysis. A typical population response induced by nicotine is seen in Fig. 3A. Since this population response is the subject of our analysis, it is important to rule out certain possible artifacts associated with the usage of a nonratiometic dye such as Oregon Green BAPTA-1. One possible artifact is that the increase in the fluorescence is related to volume changes in the axons, rather than an increase in free calcium concentration, following nicotine application. To test for this possibility, we loaded axons with Oregon-Green conjugated to dextran but lacking BAPTA-1, rendering the dye insensitive to calcium. If the increase in fluorescence in Oregon-Green-BAPTA-1 is related to volume changes in the axons, then we should see the same fluorescence increase when the nonBAPTA dye is used. This, however, was not the case (Fig. 3). We thus concluded that the fluorescence increase is related to an increase in axonal free calcium concentration.

fig. 2.

Pseudo-color calcium images of a single axon before and after nicotine application. A and B: raw data images. C and D: computed ΔF/F images from A and B. P8 nerves. Nicotine images were obtained at approximately 40 s after 50 μM nicotine application. Bar is 5 μm.

fig. 3.

Calcium fluorescence increase induced by nicotine is not due to volume changes. A: nicotine-induced fluorescence changes in axons measured with calcium sensitive dye (Oregon-Green-BAPTA-dextran) and calcium insensitive dye (Oregon-Green-dextran). B: average results from A. ΔF/F0 in calcium sensitive and insensitive dye is 0.177 ± 0.021 (n = 4) and 0.012 ± 0.001 (n = 4), respectively (P < 0.001). P6 nerves.

Next we examined the nature of the axonal calcium elevation induced by nicotine. We selectively stained large axon populations in mouse optic nerves at various stages of myelination and simultaneously measured CAPs and axonal calcium signals. Figure 4A shows a single CAP (top) and the associated axonal calcium influx (bottom). The action potential is approximately 4 ms long (as measured from the half-width of the positive deflection), while the calcium transient is much longer, being approximately 10-20 s. Our previous study (Sun and Chiu 1999) showed that this calcium transient is due to calcium influx with partial contribution from N-type calcium channels. To unambiguously demonstrate direct calcium influx mediated by neurotransmitter receptors (i.e., nAChR), we first needed to eliminate calcium influx through voltage-gated ion channels, achieved by adding the wide spectrum calcium channel blocker Cd2+ (50 μM) to the bath solution. The efficacy of calcium channel block was verified by the abolishment of the calcium transient (Fig. 4B, bottom) evoked by a single action potential (Fig. 4B, top). Next, TTX was added to block Na channels since they are slightly calcium permeable; the efficacy of the Na channel block was verified by a complete block of the action potentials (Fig. 4C). Having blocked voltage-gated ion channels that might contribute to calcium influx, we observed that bath application of nicotine (50 μM) produced a large increase in the axonal calcium level (Fig. 4D). To verify that this calcium response is receptor mediated, we applied various nAChR antagonists including d-tubocurarine (curare), mecamylamine, and α-BTX (Fig. 5A). In these experiments, the nerve was first treated with a 100-s exposure to 50 μM nicotine to elicit an axonal calcium response, which was followed by 40-60 min of antagonist application before the same concentration of nicotine was applied for a second time. Curare markedly (20 μM) reduced the second response by 87% relative to the first response (Fig. 5, A and B) in neonatal nerves. The nicotine-induced calcium response is much smaller in adult nerves, but the response is still inhibited by curare (Fig. 5D). In control experiments, no reduction in the second nicotine response was seen if curare was absent in the 40- to 60-min intervening period between the first and second nicotine applications, showing that the reduction in the second nicotine response in curare was not due to receptor desensitization. Similarly, the nicotinic receptor antagonist mecamylamine (10 μM) inhibited the nicotine response (Fig. 5, A and E) in neonatal nerves. In contrast, α-BTX (100 nM, 60-min application) is without effect on the nicotinic response (Fig. 5, A and D). Since α-BTX has a MW of 8,000, which is considerably larger than the MW of 681 for curare, the possibility existed that α-BTX may not be gaining access to the receptors due to restricted diffusion in the extracellular space. To test this possibility, we tested accessibility of another high MW toxin, α-DTX (MW = 7,054) in P7 optic nerves. Since DTX blocks delayed rectifying K channels in optic nerves and prolongs the action potential (Devaux et al. 2002), toxin accessibility can be determined from the broadening of the action potential after the K channels are blocked. We found that α-DTX produced a dramatic prolongation of the action potential that reached a steady state at approximately 15 min after toxin application (data not shown), proving that axonal receptors are accessible to toxins with MWs comparable to α-BTX.

fig. 4.

Demonstration of nicotine-mediated calcium signaling in axons of P7 optic nerve. A-C: simultaneous recordings of single action potential (top) and evoked axonal calcium transients (bottom). D: response of resting axonal calcium to nicotine with voltage-gated Ca and Na channels blocked. Axonal populations are selectively stained with calcium indicators (see methods). Concentration of Cd2+ is 50 μM and TTX is 1 μM.

fig. 5.

Effects of nicotinic acetylcholine receptor (nAChR) antagonists on nicotine-mediated axonal calcium response. A: average histograms for 3 antagonists for neonatal nerves (P2-P3). In these studies, a 1st nicotine (50 μM) response was elicited, and nicotine was washed away while antagonists (20 μM for curare (n = 4), 100 nM for α-BTX (n = 4), and 10 μM for mecamylamine (MCA; n = 1) were applied for 40-60 min before a 2nd nicotine response was elicited from the same nerve. In control studies, the 2nd nicotine response not reduced relative to the 1st one if antagonists were omitted between the 1st and 2nd nicotine response. B and D: representative axonal calcium responses at P2 and P3 in curare and α-BTX. C: nicotine response for a P31 nerve before and after curare application. E: inhibition by mecamylamine. ***P < 0.001.

From the antagonist profile (Fig. 5A), the nicotine receptors in neonatal mouse optic nerves are certainly not pure α7 subtype, since a pure α7 homomer would be very sensitive to α-BTX. Endogenous nAChRs could be hetero-multimers of various subunits, including the β subunit. To further assess the subunit composition in the neonatal mouse optic nerves, we examined the agonist profile of the axonal calcium response. Since β subunits have been suggested to determine nAChR sensitivity to cytisine (Luetje and Patrick 1991), we examined the sensitivity of the calcium response to cytisine, a nicotinic agonist. Figure 6A shows that when tested at two concentrations (1 and 10 μM), the rank order of potency for various agonists is (most potent first) cytisine > nicotine >> acetylcholine. Since it has been suggested that the β4 subunit confers cytisine sensitivity and β2 subunit confers cytisine insensitivity, our results are consistent with the presence of β4 subunits in the neonatal mouse optic nerves. However, the precise subunit composition of functional AChRs in the optic nerves remains open. Finally, the nicotinic response was abolished when nicotine was applied in a calcium-free bath solution, demonstrating that the bulk of the nicotinic response was due to calcium influx (to be discussed in Fig. 9).

fig. 6.

Effects of various neurotransmitters on axonal calcium response in neonatal nerves. A: effects of 3 nAChR agonists tested at 1 and 10 μM on the axonal calcium response in P3-P4 nerves. Rank order of potency is cytisine > nicotine >> acetylcholine. Axonal calcium response at 1 μM is cytisine = 4.7 ± 0.8% (n = 8); nicotine = 4.4 ± 0.8% (n = 8); and acetylcholine = 1.4 ± 0.4% (n = 8). Differences between cytisine, nicotine, and acetylcholine responses are statistically significant (**P < 0.01). Response at 10 μM is cytisine = 19.8 ± 1.9% (n = 8); nicotine = 16 ± 1.6% (n = 8; **P < 0.01), and acetylcholine = 2.4 ± 0.4% (n = 8; ***P < 0.001). B: axonal calcium response: nicotine (57 ± 2%, n = 12; 50 μM), kainate (15 ± 3%, n = 5; 200 μM), BzATP (3.5 ± 0.4%, n = 4; 200 nM), and ATP (7 ± 1.5%, n = 4; 0.3 mM). Differences between nicotine and the other neurotransmitters are statistically significant (***P < 0.001). All experiments from P3-P4 nerves.

fig. 9.

Effects of calcium-free solutions on the nicotine response in neonatal nerves. Compound action potential amplitudes (top rows) and axonal calcium signals (bottom row) were measured in a P7 optic nerve in response to nicotine (100 s, 50 μM). Nicotine was sequentially applied in normal Ringer (NR), in calcium-free Ringer solution, and in NR. The nerve was allowed to equilibrate for 40 min in each type of Ringer solution before nicotine was applied. A single action potential was evoked every 30 s throughout the experiment. Arrow (bottom rows, left) points to a calcium transient evoked by a single action potential. Nicotine depressed action potential amplitude in the presence or absence of bath calcium, while measured axonal calcium response was largely, but not completely, abolished by removal of calcium from perfusate.

Kainate and ATP

Glial cells are known to contain neuroactive substances (Martin 1992), and it is possible that nicotine stimulates glial cells to release these substances, which diffuse to axons to induce a calcium response. We examined two likely candidates released from glial cells, glutamate and ATP. For glutamate, we used the analog kainate known to activate calcium permeable channels. For ATP analogs, we used both BzATP and ATP. Figure 6B shows that none of these neuroactive substances, when examined in their respective effective concentrations, evoked axonal calcium responses that approached that seen with 50 μM nicotine. Hence, the simplest explanation for the nicotine-induced calcium response is that it results from direct activation of nAChR on axons.

Effects of nicotine on the CAP

In addition to evoking axonal calcium influx, nicotine also causes an abrupt reduction of the CAP in the neonatal mouse optic nerves. Figure 7A shows that, during a maintained nicotine application at 50 μM, the action potential amplitude first undergoes a transient depression and then recovers to a sustained, depressed level. This depression is dose dependent, with the half-maximal inhibition of the action potential occurring at approximately 2-5 μM nicotine. Figure 7B shows representative CAP traces before and at 2 and 10 min during nicotine application. The transient nature of the depression presumably reflects receptor desensitization. The transient depression of the action potential was largely prevented when nicotine was applied to nerves pretreated with curare (Fig. 8A). The nature of the sustained depression of the action potential by nicotine in the presence of curare is unclear; it could be due to a nonspecific action of nicotine in the mouse optic nerves. The inhibitory effect of nicotine (50 μM) on the action potential was also prevented by mecamylamine (Alkondon and Albuquerque 1993) at 10 μM (Fig. 8D). In comparison, methyllycaconitine (MLA) at 5 μM was only partially effective (Fig. 8B). In contrast, dihydro-β-erythroidine (DHβE) was completely ineffective between 40 nM and 20 μM (Alkondon and Albuquerque 1993; Rogers et al. 2001). As will be mentioned in the discussion, this antagonist profile provides strong argument against glial nAChRs mediating the inhibitory effects on the action potentials.

fig. 7.

Nicotine depresses action potentials in neonatal optic nerves. Compound action potentials were measured before and after bath application of nicotine. A: dose-dependent effect of nicotine on the action potential amplitude during maintained nicotine application. B: representative compound action potential traces before and at 2 and 10 min during nicotine application.

fig. 8.

Effects of antagonists on the nicotine-induced depression of action potentials. A: curare (20 μM) partially prevented the depression of action potentials induced by nicotine. P6 optic nerve. B: 5 μM MLA is partially effective. C: DHβE ≤20 μM is ineffective. D: 10 μM mecamylamine (MCA) completely prevents nicotine from depressing action potentials.

The reduction of action potentials suggests that nicotine depresses axonal excitability. What is the mechanism by which nicotine depresses axonal excitability? There are at least two possibilities. First, the nicotinic receptor is permeable also to Na and K, and activation of nonspecific cationic permeability on axons would lead to membrane shunting, thereby reducing the amplitude of the action potential. Second, the axonal calcium influx induced by nicotine could cause a change in membrane conductance that could block excitability (e.g., either by activating a calcium-activated K conductance or by inhibiting voltage-gated Na conductance). To sort out these two possibilities, we performed simultaneous measurement of action potentials (Fig. 9, top) and axonal calcium signals (Fig. 9, bottom) with or without calcium present in the bath solutions. In calcium-free Ringer solutions, we replaced calcium with magnesium to maintain a constant extracellular divalent cation concentration to avoid changes in surface charge effects and added 1 mM EGTA. Figure 9 (left column) shows that, with calcium present in the Ringer solution, a brief application of nicotine causes a transient depression of the action potential and a corresponding transient rise in axonal calcium; the two effects are mirror images of each other. Each action potential (generated at 1/min) evoked a calcium spike (arrow) resulting from calcium influx mediated partly through N-type calcium channels (Sun and Chiu 1999). We then applied calcium-free Ringer solutions. This caused a slight reduction in the CAP (data not shown). However, when nicotine was added, there was a similar reduction in the action potential, but without any parallel increase in the axonal calcium (Fig. 9, middle). This shows that the nicotine-induced calcium response is due to calcium influx. Furthermore, the calcium influx is not responsible for reducing the action potential. The effect is fully reversible on re-introducing calcium in the Ringer solution (Fig. 9, right). Taken together, these results show that nicotine mediates at least two independent events on the optic nerve axons: induction of axonal calcium elevation (via calcium-permeable nAChR channels) and inhibition of axonal excitability (via membrane shunting due to the nonspecific cationic permeability of the nAChR channels).

Since external calcium has been shown to potentiate the nAChR response in various tissues (Liu and Berg 1999), we performed a more detailed analysis on possible calcium dependence of the effect of nicotine on action potentials using long nicotine exposure (Fig. 10). It has been shown that, in the absence of extracellular calcium, Na influx through the nAChRs under physiological conditions is significantly reduced (Liu and Berg 1999). If the axonal nAChRs are similarly regulated, we should expect less action potential reduction in calcium-free Ringer solution in response to nicotine. Figure 10, A and C, shows that this is not the case; indeed, external calcium actually lessens the inhibitory effect of nicotine (Fig. 10C). Furthermore, the rate of desensitization (reflected by the return of the action potential amplitude to the normal, prenicotine level) is not affected by calcium (Fig. 10B). Thus axonal nAChRs appear not to be regulated by extracellular calcium.

fig. 10.

Axonal nAChRs in the neonatal nerves are not potentiated by external calcium. A: effects of external calcium on the nicotine-induced depression of the action potentials. Both optic nerves from the same mouse were dissected, and 1 nerve used for the 2.4-mM calcium study and the contralateral nerve for the 0-mM calcium study. A total of 5 mice were used. B: comparison of the rates of desensitization by normalizing the averaged response of the 2.4 and 0 calcium trace from A at the peak depression. C: bar chart of the minimum action potential amplitude during nicotine application from A. P7, n = 10 nerves.

nAChR elevates axonal [Ca2+]i without using voltage-gated calcium channels

How much of the nicotine-induced calcium influx is attributed to calcium influx via voltage-gated calcium channels? Calcium channels might contribute to calcium entry if nAChR activation causes a membrane depolarization. In certain mammalian neurons, nAChRs elevate [Ca2+]i primarily by promoting calcium influx through calcium channels; the calcium influx component directly through nAChR is very small (Barrantes et al. 1995). Is the neuronal model applicable to axons? To address this issue, we performed the experiment shown in Fig. 11A. We first depolarized the axons with a 12-mM K Ringer solution, which elevated [Ca2+]i due presumably to activation of voltage-gated calcium channels (Fig. 11A, bottom). This membrane depolarization was associated with a significant depression of the action potentials as shown (Fig. 11A, top). The nerve was washed for 10 min and followed by nicotine (50 μM) application. This depresses the action potential to a similar level as the K-solution (Fig. 11A, top), suggesting equivalent membrane depolarization and comparable activation of calcium channels in both cases. However, the observed [Ca2+]i elevation in nicotine is approximately 10 times larger than that observed in the K-depolarization (Fig. 11A, bottom), suggesting negligible contribution of voltage-gated calcium channels to the nicotine-induced calcium response.

fig. 11.

Nicotine does not elevate axonal calcium by promoting voltage-gated calcium channel activation. A is from mouse; B and C are from rat. A (top): high K+ (12 mM for 100 s) and nicotine (50 μM for 140 s) produced similar reduction of compound action potentials (to ∼10%) in a P3 mouse optic nerve. A (bottom): corresponding resting axonal calcium. B: grease-gap recording of compound resting membrane potential from P7 rat optic nerve. Recorded pretreatment potentials ranged from -25 to -35 mV. Application of nicotine (50 μM) did not result in any consistent change in resting potential. Application of GABA as a control induced a consistent depolarization in all nerves studied (n = 4) as expected for neonatal specimens (Sakatani et al. 1992). C: depression of compound action potential in P7 rat optic nerve by 50 μM nicotine (left) and 1 mM GABA (right). Smaller action potential in each panel was obtained approximately 50 s after bath application of the agents; larger action potential represents control before application. GABA and nicotine depressed the action potential to 15 ± 0.05% (n = 3) and 62.6 ± 0.04% (n = 3; P < 0.002), respectively, in P7 rat optic nerves.

One uncertainty of this study is that the reduction in action potential in nicotine may be due to membrane shunting rather than depolarization. We therefore directly measured membrane potential using a grease gap during nicotine application. Since neonatal mouse optic nerves are too small to be mounted in the gap, we switched to the larger rat neonatal nerves (also at P7) for this purpose. We first verified that P7 rat optic nerves displayed a nicotine-evoked axonal calcium response similar to that seen in P7 mice (data not shown). Interestingly, when P7 rat optic nerves were mounted in the grease gap, no membrane depolarization was observed when nicotine was bath applied (Fig. 11B, n = 4). In contrast, GABA (1 mM) did cause a membrane depolarization as expected (Sakatani et al. 1992), demonstrating that our technique can detect neurotransmitter-mediated depolarization. Figure 11C shows that 1 mM GABA depressed the action potential to a larger degree than 50 μM nicotine. It is possible that the depolarization in nAChR is small and escaped our detection. Alternatively, the shunting produced by the increase in Na and K permeability due to nAChR activation is not sufficient to override the resting conductance to significantly depolarize the membrane. From the studies in Fig. 11, we conclude that the elevation in axonal [Ca2+]i evoked by nAChR activation is not accompanied by significant membrane depolarization and that contributions from voltage-gated calcium channels are negligible. In most of the experiments in this paper, nicotine was applied without calcium channel blockers.

Calcium-induced calcium release

To examine if calcium-induced calcium release (CICR) contributes to the axonal calcium elevation induced by nicotine, we blocked microsomal calcium pumps with bath application of thapsigargin (2 μM) for 30 min (Sharma and Vijayaraghavan 2001). At the end of the application, the CAP was not affected. We did no see any significant effect of thapsigargin on the nicotine (50 μM)-evoked calcium response (data not shown, n = 2). These preliminary studies therefore suggested that CICR is not important in the nAChR-mediated axonal calcium elevation.

Relationship between nAChR expression and myelinogenesis

Mammalian optic nerves express GABAA receptors whose expression is downregulated with myelin maturation (Sakatani et al. 1992). We therefore examined whether there is a similar decline of nicotine response in mouse optic nerves during development. Figure 12B shows a decline of the inhibitory effect of nicotine on the CAPs during myelinogenesis. During the premyelinated stage (P1-P3), nicotine exerts a strong inhibition on the action potential, reducing it by approximately 90%. As myelin is gradually formed, nicotine becomes progressively less effective in reducing action potentials, so that by P15, the inhibitory effect of nicotine on the action potential is barely noticeable. In parallel, calcium imaging reveals a downregulation of the nicotine-induced calcium response during development (Fig. 12A). An interesting question is whether the nicotinic response is expressed in dysmyelinated axons. We therefore examined the CNS dysmyelinating mutant mice Jimpy. At P17, a developmental time point in normal mouse optic nerves when the nicotine-induced calcium response is reduced to approximately 10%, we still detected significant nicotine-mediated calcium response, at approximately 50%, in the dysmyelinating mutant. Dysmyelinating axons therefore express nAChRs in the absence of normal myelin.

fig. 12.

Developmental changes in nicotine sensitivity in optic nerves. A: developmental changes of nicotine-induced (50 μM) calcium response from P1 to P31. Solid bars, normal mice; stripe bar, dysmyelinating mutant (Jimpy). Axonal ΔF/F is P1: 0.954 ± 0.228 (n = 5); P3: 0.521 ± 0.068 (n = 6); P4: 0.41 ± 0.072 (n = 5); P5: 0.241 ± 0.055 (n = 5); P6: 0.197 ± 0.039 (n = 6); P7: 0.212 ± 0.047 (n = 5); P15: 0.11 ± 0.01 (n = 4); P31: 0.048 ± 0.017 (n = 4); and P17 (Jimpy): 0.518 ± 0.126 (n = 4). B: developmental changes of nicotine-induced (50 μM) reduction in compound action potentials. Action potentials were reduced by 93.8 ± 3.4% (P1; n = 5), 83.9 ± 5.6% (P3; n = 6), 55.4 ± 2.1% (P6; n = 6), 48.7 ± 3.9 (P7; n = 5), and 3.9 ± 1.5 (P15; n = 5).

Activity-dependent release of acetylcholine

We next examined if axonal nAChRs are activated during normal nerve activity. If acetylcholine is released during nerve activity and activates axonal nAChRs, there should be a reduction of the CAP (Fig. 7). Furthermore, any inhibitory effect of nAChR activation on the action potential should be potentiated if the action of acetylcholine is prolonged with neostigmine (a blocker of the enzyme acetylcholinesterase that normally degrades extracellular acetylcholine) or attenuated if the nAChRs are blocked by curare. We used this rationale to design the experiment in Fig. 13. In the control (normal Ringer solution), we applied a tetanus (2 min, 5 Hz) and measured the amplitude of the CAPs during the tetanus. The action potential shows a gradual decline during the tetanus (Fig. 13A, control). There are several traditional factors that might contribute to this action potential decline, including potassium accumulation and ion channel inactivation. However, a new factor we are interested in is whether nAChR activation might contribute to this decline. To test this idea, we applied neostigmine (0.2 μM) to the contralateral optic nerve from the same animal to prolong the action of any released acetylcholine. In these studies, both optic nerves were dissected from the same mouse and equilibrated for 1 h in oxygenated Ringer solution before studies began. Each nerve (control or drug treated) was subjected to only one tetanus. Neostigmine has no effect on the shape and amplitude of a single compound action potential (data not shown). However, neostigmine significantly deepened the inhibition of the action potential during a tetanus (Fig. 13, A and C). This neostigmine-sensitive component of the action potential decline is eliminated when curare (20 μM) was co-applied to block nAChRs (Fig. 13, B and C). Interestingly, neostigmine still deepened the action potential inhibition during the tetanus when calcium was omitted from the bath, suggesting that activity-dependent acetylcholine release is calcium independent (Fig. 13C). These experiments suggest that acetylcholine is released during nerve activity, targets axonal nAChRs, and causes a progressive shunting of the membrane potential and depression of excitability during high-frequency stimulation.

fig. 13.

Activity-dependent activation of nAChRs in neonatal optic nerves. A: action potential amplitudes during 5-Hz stimulation for 2 min with or without 0.2 μM neostigmine (n = 11). B: 5-Hz stimulation with curare (20 μM) co-applied with neostigmine (n = 12). C: action potential amplitudes at the end of 2-min stimulation at 5 Hz. Data from A and B. Amplitude of action potential in normal Ringer solution at the end of 2-min stimulation was taken as control, i.e., 100%. Compared with this control, action potential amplitude was 80 ± 2% (n = 11, *P < 0.05) in neostigmine and 112 ± 2% (n = 12, P > 0.05) in curare plus neostigmine. In 0-mM calcium-free Ringers, action potential amplitude in 0.2 μM neostigmine was 85 ± 2% compared with control (n = 10, *P < 0.05). Thus calcium-free solutions did not prevent deepening of action potential inhibition by neostigmine.


Significance of this study

Despite the extensive work already done on acetylcholine signaling in the visual pathways (Brocher et al. 1992; Butt et al. 2000; Edwards and Cline 1999; Imamura and Kasamatsu 1989; Nobili and Sannita 1997; Reed et al. 2002; Swanson et al. 1987), little is known regarding nAChR expression on the axon proper of the retinal ganglion cells. Here, we utilize a method developed in our laboratory to selectively stain axon populations with calcium indicators, allowing us to observe directly nicotine-mediated calcium influx in axons, examine receptor antagonist profile, correlate calcium signaling with nerve excitability, and assess the functionality of nicotine receptors in the mammalian optic nerves. By cutting the ganglion cell bodies and the presynaptic terminals, nAChR expression on the axon proper is studied in complete isolation from any synaptic elements.

Expression of nAChRs on axons

Several pieces of evidence suggest that nAChRs are present on axons of neonatal optic nerves. First, nicotine application induced an axonal calcium influx that is blocked by nAChR blockers α-tubocurarine and mecamylamine. Interestingly, the nAChR is insensitive to 100 nM of α-BTX, suggesting that the axonal nAChR is not a pure α7 subtype. The antagonist profile of axonal nAChR is consistent with patch-clamp studies demonstrating that nAChRs on the soma of mammalian retinal ganglion cells are sensitive to α-tubocurarine but insensitive to α-BTX (Lipton et al. 1987). Hence, the same nAChR subtype appears to be distributed on both the soma and the axon. Second, bath application of nicotine causes an abrupt reduction in the action potential, consistent with a direct shunting effect of nAChRs on axons. The restoration of action potentials during a sustained nicotine application (Figs. 7 and 8) is consistent with the known desensitization of these ligand-gated ion channels. Third, an indirect effect of nAChR activation on glial cells is rendered unlikely, as described in Indirect effects of nAChR activation on glial cells.

Indirect effects of nAChR activation on glial cells

It is possible that axonal calcium influx is mediated by activation of nAChRs on glial cells. Recent studies have established the presence of nAChRs on astrocytes (Sharma and Vijayaraghavan 2001; Teaktong et al. 2003). However, the astrocytic nAChRs are of the α7 subtype and are sensitive to α-BTX (Sharma and Vijayaraghavan 2001). O-2A progenitor cells cultured from P7 corpus callosum also express nAChRs (Rogers et al. 2001). However, nAChRs in O2A cells are blocked by the α4/β2 antagonist DHβE at 10 nM (Rogers et al. 2001), which contrasts with the DHβE insensitivity in our study at ≤20 μM (Fig. 8C). Release of a diffusible substance from glial cell is a possibility. One candidate might be potassium ions released by glia, which cause axonal depolarization, calcium channel activation, and axonal calcium influx. However, we cannot mimic the large axonal calcium influx seen in nicotine by K depolarization (Fig. 11). Other possible candidates for a diffusible substance from glial cells include glutamate and ATP. However, analogs of these substances did not elicit a comparable calcium response seen in nicotine (Fig. 6B). Thus the simplest explanation for the axonal calcium response and the abrupt reduction in action potential, both blockable by d-tubocurarine and mecamylamine, is that they result from nAChRs directly present on axons.

Does calcium influx through nAChRs account for all the calcium elevation?

Even though nAChR is calcium permeable, this receptor elevates [Ca2+]i by different means in different cell types. In hippocampal neurons, the nAChR elevates [Ca2+]i primarily by promoting calcium influx through voltage-gated calcium channels; calcium influx directly through nAChRs contributes little to the neuronal [Ca2+]i elevation (Barrantes et al. 1995). In astrocytes, nAChR elevates [Ca2+]i primarily by CICR (Sharma and Vijayaraghavan 2001). In this study, the axonal [Ca2+]i elevation triggered by nAChR activation has virtually no contribution from voltage-gated calcium channels since nAChR activation is not accompanied by detectable membrane depolarization (Fig. 11B). Why does activation of the cation nonselective nAChR pore not lead to membrane depolarization? One possibility is that the calcium influx through the nAChR pore might activate a calcium-activated K conductance that counteracts the depolarizing action of nAChR activation. Even without membrane depolarization, nAChR activation evidently leads to significant membrane shunting via conductance increase to impact excitability. With regard to the role of CICR in nAChR-induced calcium response, we did not detect significant effect of thapsigargin on the response. A smaller contribution from internal Ca stores triggered by nAChRs, possibly very localized at the subaxolemmal region (and therefore difficult to detect with confocal microscopy) with significant effects on excitability, cannot be ruled, given the recent demonstration of CICR mechanisms in CNS axons in ischemic conditions (Ouardouz et al. 2003).

Functions of axonal nAChR

We propose that axonal nAChRs have various functions during development, including modulation of axonal transport, axonal elongation, and modulation of axonal excitability.

axonal elongation and guidance. In the developing retina, nAChR-mediated spontaneous activity may be important in neurite extension and guidance during development (Feller et al. 1996; Lipton et al. 1988). nAChR-mediated axonal calcium elevation might alter the dynamics of actin/cytoskeletal assembly (Bentley and O'Connor 1994; Lankford et al. 1996) and modulate axonal elongation during development. The source for acetylcholine might be the glial cells, because the acetylcholine synthesizing enzyme (ChAT) has been demonstrated in cultured astrocytes (Wessler et al. 1997) and in oligodendrocytes in situ (Lan et al. 1996).

regulation of axonal transport. Axonal transport of proteins is crucial to long-term maintenance of the structure and excitability of axons, and the calcium ion is an important modulator of axonal transport (Breuer and Atkinson 1988a,b; Chan et al. 1980; Kanje et al. 1982; Worth and Ochs 1982). In the cat peroneal nerve, axonal transport was inhibited in a calcium-free bath solution (Chan et al. 1980). Calcium channel blockers, such as dihydropyridines and d-600 (Breuer and Atkinson 1988b; Kanje et al. 1982), cadmium, and nickel (Bartlett et al. 1999) have been shown to modulate axonal transport under various normal and pathological conditions. Collectively, voltage-gated calcium channels present on optic nerve axons (Brown et al. 2001a,b; Fern et al. 1995; Sun and Chiu 1999), as well as calcium-permeable nAChRs described in this study, may be involved in regulation of axonal transport in optic nerves during development.

modulation of excitability. We found that low-frequency stimulation at 5 Hz, which is well within the range of spontaneous activity of 10-30 Hz in a developing retina (Masland 1977; Meister et al. 1991), is sufficient to induce acetylcholine release (Fig. 13). Glial cells are the likely source of acetylcholine since they contain choline acetyltransferase (Lan et al. 1996; Wessler et al. 1997). Activation of nAChRs might modulate excitability by depressing action potential propagation.

Pathological implications: nAChR expression in demyelinated nerves

In addition to normal nerves, we found that nAChR is expressed in dysmyelinated axons from the mutant Jimpy mice (Fig. 12A). The expression of nAChR in demyelinated axons is particularly interesting for several reasons. First, given that acetylcholine is known to have a role in developmental plasticity, the existence of nAChR on demyelinated axons is functionally important in axon biology during and following demyelination. Second, an upregulation of axonal nAChR expression, as suggested by Fig. 12A, may be relevant to calcium-mediated toxicity. Glial cells or inflammatory cells near sites of nerve lesions may provide an abnormal source of acetylcholine in pathological conditions. Furthermore, axonal degeneration is a major factor in the pathology of multiple sclerosis (Trapp et al. 1998), and an important issue is whether expression of nAChRs on demyelinated axons might contribute to excitotoxic calcium elevation. Molecular pathways that mediate calcium influx into demyelinated axons include voltage-gated calcium channels, Na/Ca exchangers, and receptor-mediated calcium influx (nAChRs). These molecular pathways for calcium influx may be dysregulated in pathology, possibility contributing to calcium-mediated axonal damage in demyelinating diseases. An example is the recent finding that axonal N-type calcium channels appear to be upregulated in lesion sites in CNS white matter from patients with multiple sclerosis (Kornek et al. 2001), leading the authors to suggest a possible link between axonal damage and excessive calcium channel expression. Likewise, a possible linkage between axonal nAChR expression and axonal pathology in demyelinating diseases is an interesting area for further investigation.



Work in S. Y. Chiu's laboratory is supported by Grants RO1-23375 from the National Institutes of Health (NIH) and RG-3247-A-6 from the National Multiple Sclerosis Society underwritten by the Estate of Norman Cohn. P. K. Stys is supported by a Career Investigator Award from the Heart and Stroke Foundation of Ontario and in part by NIH Grant R01 NS-40087-01.


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