ACh has been shown to affect numerous functions in developing neurons in vitro, including neurite outgrowth, growth cone guidance, synapse formation and synaptic transmission (Zheng et al., 1994; Lauder and Schambra, 1999; Woodin et al., 2002; Rudiger and Bolz, 2008). Here, we describe a role for cholinergic modulation at the growth cone, where ACh affects an important component of growth cone motility, namely filopodial dynamics. We report that ACh induced a transient but significant filopodial elongation in B5 neurons of the buccal ganglion of Helisoma trivolvis. The signaling cascade resulting in filopodial elongation is via opening of nAChRs, a depolarization of the membrane potential, and a subsequent elevation of [Ca]i in growth cones. Moreover, by performing experiments on physically isolated growth cones, we demonstrated that ACh can act as a local signal at the growth cone to elongate filopodia.
ACh and nAChRs
nAChRs appeared to be both necessary and sufficient for ACh to elicit its effects on growth cone filopodial dynamics, intracellular calcium, and neuronal electrical properties. Treatment with DMPP, a nonselective nAChR agonist, fully mimicked the effects of ACh on membrane depolarization, reduction in Rin, elevation of [Ca]i, and filopodial elongation. Meanwhile, TC, a well-known antagonist of nAChRs, completely inhibited all DMPP-induced responses. These results not only supported the specificity of our pharmacological approach, but also provided convincing evidence that ACh acted through activation of nAChRs to produce these effects. Taken together, our results add a novel role to the critical functions of nAChRs in cholinergic modulation of cellular properties in both vertebrates and invertebrates (Fu et al., 1998; Clementi et al., 2000; Woodin et al., 2002; Cobb and Davies, 2005).
nAChRs belong to a large Cys-loop family of ligand-gated ion channels including the 5-HT3, glycine, and GABAA receptors (Le Novere and Changeux, 1995). nAChRs exist as pentameric complexes assembled either from five copies of a single subunit or by a composition of five different subunits (Clementi et al., 2000). Although the molecular identities of nAChRs are well studied in mammalian systems, much less information is available in gastropods. Van Nierop and Smit identified nAChR subunits in the pond snail Lymnaea stagnalis, a closely related species (van Nierop et al., 2005; van Nierop et al., 2006), and cloned a total of twelve subunits of nAChR. Considering that ACh treatment in this study caused the depolarization of RMP and an elevation in [Ca]i in Helisoma B5 neurons, it is likely that the effect of ACh was contributed by the classic, cation-selective nAChRs. A characterization of Helisoma nAChRs at the molecular level is beyond the scope of this study but will extend our understanding of cholinergic modulation in the Helisoma nervous system in the future. Because the effects of ACh on growth cones and electrophysiological properties could be fully mimicked by the nAChR agonist, DMPP, we did not pursue any potential involvement of muscarinic ACh receptors in regulating growth cone filopodial length and [Ca]i.
ACh and Cell Excitability
Cholinergic modulation of cell excitability has been studied in some detail in gastropods. Salivary gland cells in Helisoma respond to ACh by a long-lasting depolarization followed by hyperpolarization (Bahls, 1987). Moreover, ACh is thought to be the core neurotransmitter to control neuronal activity in the feeding central pattern generator in Lymnaea, such as protraction phase premotor interneurons N1L and N1M (Elliott and Vehovszky, 2000). Cholinergic projections to the Lymnaea proesophagus modulate foregut contractile activity (Perry et al., 1998). Furthermore, ACh acts on an ionotropic receptor sensitive to nicotinic antagonists to evoke an afterdischarge in Aplysia bag cell neurons (White and Magoski, 2012).
We demonstrated that ACh induced rapid and significant changes in electrical activity in B5 neurons. Both treatment with ACh and the nAChR agonist, DMPP, caused a reduction in Rin in a dose-dependent manner, suggesting the opening of nAChRs. The activation of nAChRs, in turn, induced the depolarization of the membrane potential. Relatively lower doses of ACh or DMPP resulted in an increase in spiking frequency, whereas higher concentrations caused cell silencing at a depolarized membrane potential.
ACh, Ca, and Growth Cone Motility
We report that both stimulation with ACh and activation of nAChRs by DMPP resulted in a quick and pronounced increase in [Ca]i in growth cones. Interestingly, the elevation of [Ca]i and the increase in filopodial length occurred with a very similar time course. Considering the critical role of spiking activity in regulating [Ca]i within neurons (Spitzer, 2006), and that causality has been demonstrated between an elevation in [Ca]i and filopodial elongation in B5 neurons (Rehder and Kater, 1992), the present study is consistent with the hypothesis that ACh functions through an increase in [Ca]i to elongate growth cone filopodia. After the replacement of culture medium with a Ca free solution, both the filopodial elongation and the elevation of [Ca]i induced by ACh were blocked, suggesting that Ca influx plays a critical role in ACh-signaling at the growth cone. In fact, other neurotransmitters and neuromodulators have been demonstrated to signal through [Ca]i to regulate growth cone functions (Henley and Poo, 2004; Gomez and Zheng, 2006). For example, the gaseous messenger nitric oxide affects growth cone filopodial dynamics in Helisoma B5 neurons via a Ca-dependent mechanism (Van Wagenen and Rehder, 2001; Trimm and Rehder, 2004; Welshhans and Rehder, 2005; Welshhans and Rehder, 2007). An increase in [Ca]i is able to reduce neurite outgrowth rate in Helisoma neurons (Cohan, 1992). In addition, [Ca]i is required for glutamate, netrin-1, and myelin-associated glycoprotein to guide growth cone turning in cultured Xenopus spinal neurons (Zheng et al., 1996; Ming et al., 2001). Upon the elevation of [Ca]i at the growth cone, multiple Ca-mediated signaling pathways could be activated to translate external signals into cytoskeletal changes. Our lab previously showed that calmodulin and the Ca-dependent phosphatase calcineurin are acting downstream of Ca to elongate filopodia (Cheng et al., 2002). Moreover, calmodulin-dependent protein kinase II is found to mediate ACh-induced chemoattraction in growth cone guidance (Zheng et al., 1994).
ACh has been shown to regulate cell movements, cell proliferation, and neuronal differentiation in various developing central nervous systems (Lauder and Schambra, 1999). Although the roles played by ACh in neuronal development in vivo are yet unclear, evidence for a functional role of ACh in developing neurons came from the studies of cultured embryonic Xenopus spinal neurons (Zheng et al., 1994). ACh was found to act as a chemoattractive guidance cue that elicited growth cone turning behavior towards the source of ACh release. Here, we extended this research by studying the effect of ACh on growth cone filopodia. Filopodia on growth cones are essential for growth cone guidance, and filopodial elongation increases the area that a growth cone can sample during pathfinding (Kater and Rehder, 1995; Rehder et al., 1996). A transient elongation of filopodia, as seen in this study in response to stimulation with ACh, could play a critical role in decision-making at the growth cone. Longer filopodia would encounter cues located 10–20 μm ahead of the advancing growth cone proper, could result in a change in the direction of growth towards or away from the cue depending on the signal content, and ultimately, upon contact of an appropriate cellular target, could transform a growth cone into a presynaptic structure. In our experiments, ACh was bath-applied, mimicking a general, extrasynaptic stimulation of B5 neurons. To determine the location at which ACh acted to elicit filopodial elongation, we physically isolated growth cones and demonstrated that they responded to ACh treatment in a similar fashion as intact growth cones did, suggesting that ACh can regulate growth cone motility at the growth cone proper. We did not attempt to produce gradients of ACh across growth cones to determine if this would result in asymmetrical filopodial elongation, as might be expected to precede growth cone turning. In fact, filopodial asymmetry on growth cones of Xenopus neurons was found to precede growth cone turning in responses to glutamate gradients (Zheng et al., 1996). Additionally, our lab reported that both transient changes of growth cone filopodial dynamics (Van Wagenen and Rehder, 1999) and decreases in nerve growth speed (Trimm and Rehder, 2004) can be triggered by nitric oxide, a phenomenon we described as “slow-down and search” behavior. Here, the ACh-induced change in filopodial dynamics may serve as a first response of an extending neurite to ACh encountered during pathfinding.
Although the sources of ACh release and physiological concentrations reached in the Helisoma buccal ganglia are unknown, several studies in which ACh release was measured suggest that the concentrations used in our in vitro study are comparable to in vivo conditions. The ACh concentration detected in the vicinity of magnocellular basal forebrain neurons using the nAChR-rich patches prepared from rat myotubes as focal ACh sensors was between 480 nM to > 50 μM (Allen and Brown, 1996). This concentration range of ACh matched the concentrations used in the current study and resulted in significant electrophysiological and morphological responses in B5 neurons. B5 neurons are responsive to ACh and are themselves cholinergic (Haydon and Zoran, 1989). In vitro, the site of ACh release in Helisoma neuron B5 is thought to be mainly confined to the distal neurites, but rarely detected from the soma. ACh release from neurites and growth cones has been reported in different neurons (Hume et al., 1983; Allen and Brown, 1996; Zakharenko et al., 1999; Yao et al., 2000), suggesting that growing cholinergic axons might influence other extending axons expressing nAChRs to regulate their developmental status. Moreover, ACh has been reported to act in an autocrine fashion on presynaptic terminal of Xenopus motoneurons (Fu et al., 1998), suggesting the possibility that ACh release from axonal terminals might directly affect growing axons via an autoreceptive feedback mechanism.
In conclusion, our results provide novel insights into the effects of ACh on developing neurons. ACh affected growth cone filopodial dynamics through the activation of TC-sensitive nAChRs by depolarizing membrane potential and increasing [Ca]i. While such effect could be caused by presynaptic stimulation, supporting the well-known literature of effects of electrical activity on neurite outgrowth and growth cone motility, the finding that ACh can act locally at a growth cone suggested that ACh might also act at extrasynaptic receptors to modulate growth cone motility, neuronal pathfinding, and possibly synaptogenesis directly at the level of the growth cone.
The authors thank Dr. Chun Jiang for expert advice on the electrophysiological experiments and their analysis.