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The site at which action potentials initiate within the terminal region of unmyelinated sensory axons has not been resolved. Combining recordings of nerve terminal impulses (NTIs) and collision analysis, the site of action potential initiation in guinea-pig corneal cold receptors was determined. For most receptors (77%), initiation mapped to a point in the time domain that was closer to the nerve terminal than to the site of electrical stimulation at the back of the eye. Guinea-pig corneal cold receptors are Aδ-neurones that lose their myelin sheath at the point where they enter the cornea, and therefore their axons conduct more slowly within the cornea. Allowing for this inhomogeneity in conduction speed, the resulting spatial estimates of action potential initiation sites correlated with changes in NTI shape predicted by simulation of action potentials initiating within a nerve terminal. In some receptors, more than one NTI shape was observed. Simulations of NTI shape suggest that the origin of differing NTI shapes result from action potentials initiating at different, spatially discrete, locations within the nerve terminal. Importantly, the relative incidence of NTI shapes resulting from action potential initiation close to the nerve termination increased during warming when nerve activity decreased, indicating that the favoured site of action potential initiation shifts toward the nerve terminal when it hyperpolarizes. This finding can be explained by a hyperpolarization-induced relief of Na+ channel inactivation in the nerve terminal. The results provide direct evidence that the molecular entities responsible for stimulus transduction and action potential initiation reside in parallel with one another in the unmyelinated nerve terminals of cold receptors.
Sensory encoding in the peripheral nerve terminals of sensory neurons comprises two distinct processes (Katz, 1950; Hodgkin, 1938): an initial transduction of the stimulus energy into a receptor current and the subsequent transformation of this current into an action potential discharge. For cold-sensitive neurons, transduction is likely to involve several molecular entities including the menthol-sensitive, cooling-activated transient receptor potential melastatin 8 (TRPM8) channel (McKemy et al. 2002; Peier et al. 2002; Reid et al. 2002), heat-activated two-pore domain K+ channels (e.g. TREK-1 K+ channels; Maingret et al. 2000; Reid & Flonta, 2001; Viana et al. 2002) and a Na+,K+-ATPase-mediated hyperpolarizing current that is proportional to temperature (Pierau et al. 1974). The receptor current that ensues is transformed into action potentials by voltage-gated Na+ channels, the kinetics and availability of which are also affected by temperature. The extent to which the processes of transduction and transformation interact electrically within the nerve terminal determines the encoding of the stimulus and is consequently of fundamental importance to an understanding of cold receptor function.
In principle, the site at which action potentials initiate is determined by the spatial distribution of voltage-gated Na+ channels in the nerve terminal. The conventional view is that the receptive region of axonal membrane at the terminal is devoid of voltage-gated Na+ channels and therefore that the site of sensory signal transduction and the site of action potential initiation are spatially separate. However, extracellular recordings suggest that action potentials may be initiated and conducted within the unmyelinated nerve ending of Pacinian corpuscle afferents (Hunt & Takeuchi, 1962; Ozeki & Sato, 1964; Nishi & Sato, 1966) and frog muscle spindle afferents (Ito et al. 1974), and this is supported by immunohistochemical evidence of Na+ channels in the nerve terminal region of the Pacinian corpuscle (Pawson & Bolanowski, 2002). Despite the apparent presence of Na+ channels in the receptive nerve terminal membrane, action potential initiation is nevertheless thought to occur at the first node of Ranvier (Loewenstein & Rathkamp, 1958) where the Na+ channel density is high (Conti et al. 1976; Neumcke & Stampfli, 1982) and the membrane capacity is reduced by Schwann cell myelination.
In the guinea-pig cornea, cold receptors are likely to be Aδ-neurones and extracellular recording from their nerve terminals has revealed that they possess both tetrodotoxin (TTX)-sensitive and TTX-resistant Na+ channels at densities sufficient to support a regenerative Na+ current in the terminal (Carr et al. 2002). However, in the cornea, Aδ-axons lose their myelination at the limbus where they enter the cornea and have a long unmyelinated axon termination, so it is unlikely that action potentials are initiated at their first node of Ranvier. At present there is no evidence of clustering of Na+ channels at sites along the unmyelinated axons within the cornea (Black & Waxman, 2002) nor of morphological specializations that would favour action potential initiation at specific axonal sites. This leaves the question as to where action potentials are initiated in unmyelinated nerve terminals unresolved.
In the present study, the initiation site of action potentials generating ongoing nerve terminal impulses (NTIs) recorded extracellularly from cold-sensitive neurones in the guinea-pig cornea was determined using collision analysis. Collision of the ongoing orthodromic action potentials with antidromic action potentials evoked by electrical stimulation at the back of the eye was used to map the site of origin of the ongoing activity. In the majority of cold receptors, ongoing action potentials were initiated at points that mapped close to the site of recording. Consistent with this finding, differences in the shape of ongoing and electrically evoked NTIs were comparable with those predicted by computer simulation for initiation sites electrotonically close to the site of recording. Indeed, in some cases, the shape of the ongoing NTIs could be simulated by action potential initiation within half a length constant of the sensory nerve terminal. In these cases, Na+ current at the nerve ending contributed to action potential genesis demonstrating directly that the process of sensory signal transduction and the regenerative processes producing action potentials exist in parallel with one another in cold receptor nerve terminals.
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Establishing the site at which the action potential initiates within the terminal region of unmyelinated sensory axons is fundamental to an understanding of their function. Cold-sensitive axons provide a suitable model in which to examine action potential initiation in unmyelinated terminals because they have an ongoing discharge of action potentials and in the guinea-pig cornea it is possible to make extracellular recordings from their sensory nerve terminals (Brock et al. 1998, 2001). The site of action potential genesis was determined in corneal cold-sensitive receptors by establishing the interval over which ongoing action potentials collided with activity evoked by electrical stimulation of the ciliary nerves at the back of the eye. In three quarters of the receptors, the origin of activity in the time domain mapped to sites in the axon closer to the nerve terminal than they were to the site of electrical stimulation. As indicated in the Results, we believe that guinea-pig corneal cold receptors are Aδ-neurones that lose their myelin sheath close to the point where they enter the cornea. The loss of myelin will slow action potential propagation within the cornea, an effect that would move action potential initiation sites mapped in the time domain to sites closer to the site of recording at the nerve termination in the space domain (see Fig. 3). In accord with this suggestion, in ca 40% of receptors, the proximity of the initiation site to the nerve ending can be further inferred from differences between the shape of ongoing NTIs and those evoked by electrical stimulation of the ciliary nerve back of the eye (see Fig. 4). In the remaining one quarter of the corneal cold receptors, the origin of activity mapped to sites in the time domain that are closer to the site of electrical stimulation at the back of the eye than they are to the nerve terminal. Such sites are likely to be axonal branch points, which are known to occur close to the point where Aδ-axons innervating the cornea loose their myelin sheath at the corneo-scleral junction (Zander & Weddell, 1951). In these cases, we believe the ongoing action potentials are initiated in the receptive nerve terminal(s) of an axon branch and propagate by axon reflex antidromically to the recording site.
The accuracy of estimates of the site of action potential initiation determined using collision analysis would also be affected by directional asymmetry in axonal conduction velocity, with estimates being erroneously skewed towards the terminal if antidromic conduction (i.e. toward the nerve termination) were more rapid. Axonal branching is expected to exert the most prominent effect in this respect and along their course axons within the cornea display a moderate degree of branching (Zander & Weddell, 1951; Whitear, 1960; MacIver & Tanelian, 1993). Theoretically, at divergent branch points, the decrease in diameter of each daughter branch (Quilliam, 1956) may produce an impedance mismatch that would render conduction in the antidromic direction more rapid than that in the orthodromic direction (Goldstein & Rall, 1974). In absolute terms, each branch point at which the change in axonal diameter does not exceed a halving in the daughter branches would only be expected to produce a small lead of ca 100 μs for antidromically conducted action potentials (Manor et al. 1991). Functional (Waddell et al. 1989; Peng et al. 1999) and anatomical (Thiel, 1957; Banks et al. 1982; Heppelmann et al. 1990) evidence both suggest minimal tapering along myelinated and unmyelinated axons. At the axon termination, however, it would be predicted that there is a reduction in axon diameter and theoretically this could also slightly speed conduction in the antidromic direction relative to that in the orthodromic direction (Toth & Crunelli, 1998). It is not possible to account for these differences in orthodromic and antidromic conduction velocity but it is reasonable to assume that the errors in estimates of initiation site attributable to either axonal branching or tapering of the terminal portion of axon are small.
The NTI signal recorded extracellularly from nerve terminals is proportional to net membrane current, with positive- and negative-going signals representing outward and inward current respectively (Smith, 1988). The most commonly recorded NTI shape can be adequately simulated by an action potential propagating into the terminal and consists of an initial net outward current followed by a smaller net inward current (Carr et al. 2003). An NTI of this general shape results from antidromic invasion of the nerve terminal by an action potential initiated at a site that is electronically remote from the nerve terminal, such as that resulting from electrical stimulation of the ciliary nerves at the back of the eye. For ongoing NTIs with this conventional shape, the amplitude of the initial positive component is typically smaller than that of electrically evoked NTIs in the same nerve terminal (Brock et al. 2001). It was previously suggested that the smaller amplitude of ongoing NTIs resulted from the nerve terminal being depolarized during action potential invasion (Brock et al. 2001). Simulations of NTI shape indicate, however, that for action potentials initiating between approximately 0.6 and 3 length constants from the nerve terminal the positive amplitude of the NTI decreases monotonically as the site of initiation moves closer to the nerve termination (Fig. 4). We have previously reported that on average ongoing NTIs are smaller than their electrically evoked counterparts in cold-sensitive receptors in the cornea (Brock et al. 2001), which is consistent with our observation that action potential initiation often occurs very close to the site of recording at the nerve terminal.
Ongoing NTIs with an initial negative-going component (i.e. inward current) observed in some receptors are particularly interesting because simulations suggest that NTIs of this configuration arise from initiation of action potentials within about half a length constant of the nerve termination (Fig. 4). For these negative–positive NTIs, the initial net inward current is dominated by Na+ channel opening during action potential genesis and the later net outward current is produced as the action potential propagates away from its point of initiation. Collision analysis confirmed that these inverted NTIs result from action potentials that initiate very close to the nerve termination. Furthermore, within a single nerve terminal, ongoing NTIs with an inverted shape always occurred together with NTIs of conventional shape and collision mapping indicated that action potentials producing inverted NTI shapes initiated at a site closer to the nerve termination than those generating conventionally shaped NTIs (Fig. 5). The presence of multiple ongoing NTI shapes in a single axon termination indicates that there can be multiple sites of action potential initiation within the nerve terminal axon and that the point of initiation is labile, shifting from impulse to impulse. While this could be interpreted as indicating that action potential initiation can occur anywhere within the nerve terminal, collision analysis indicates that each NTI shape has a relatively narrow domain over which collision occurs (see Figs 1D, 2B and 5B), demonstrating that there are favoured sites of action potential initiation. It is possible that these sites are located at points along the unmyelinated axon where voltage-gated Na+ channels are clustered (Hildebrand & Waxman, 1983) as has recently been reported for the en passant boutons along the axons of mossy fibre neurons in the hippocampus of the rat (Engel & Jonas, 2005).
Sensory neurones with thinly myelinated (Aδ) and unmyelinated (C) axons often form a branched nerve terminal arbour in the periphery (Heppelmann et al. 1990, 1995; MacIver & Tanelian, 1993). The functional correlate of axonal branching is a structured receptive field composed of discrete regions of high sensitivity, figuratively designated ‘hot spots’ at which the threshold for evoking an action potential that propagates to the parent axon is relatively low (Bessou & Perl, 1969; Schmidt et al. 2002). The receptive fields of cutaneous cold-sensitive receptors can be composed of multiple discrete ‘cold spots’ at which cold stimuli increase nerve activity (Duclaux & Kenshalo, 1973). However, when two cold receptive spots were simultaneously cooled, the activity in the parent axon was the same as that evoked by cooling the most sensitive spot, i.e. the cold receptive spot generating the highest rate of action potential discharge occludes the activity originating at the other receptive spot. This suggests that each cold-sensitive spot is the termination of a separate axon branch and that each branch encodes action potentials autonomously. This implies that electrotonic interaction between the individual action potential encoders is small. Consistent with this suggestion, when one cold receptive spot was heated in receptors with multiple receptive spots, the ongoing activity was little affected presumably because activity originating at the other receptive sites where the temperature did not change maintained the ongoing activity (Duclaux & Kenshalo, 1973).
At the normal holding temperature (∼32°C), corneal cold receptors, like cold receptors in other tissues, have an ongoing discharge of nerve impulses that occur in a rhythmic manner, comprising both single impulses as well as short bursts of impulses at relatively fixed intervals (see Carr et al. 2003). In receptors with multiple cold receptive spots, it would be predicted that the ongoing nerve activity originates at the receptive site where the membrane potential reaches the threshold for action potential initiation first, i.e. the dominant pacemaker. In accord with this suggestion, in cornea, all the ongoing action potentials recorded appeared to originate within a single spatially restricted region of the nerve terminal arbour that was either located relatively close to the site of recording or, most likely, at the termination of a co-lateral axon branch. Furthermore, in the collision analyses, it was always possible to find a collision interval that completely occluded electrically evoked NTIs, indicating that all action potentials generating ongoing NTIs also propagated centrally irrespective of their site of origin within the nerve terminal arbour.
The cell bodies of cold-sensitive sensory neurons in dorsal root and trigeminal ganglia hyperpolarize and depolarize, respectively, during warming and cooling (Reid et al. 2002; Viana et al. 2002). On the basis of changes in NTI shape recorded at the same temperature during heating and cooling, we have previously suggested that this also occurs at the sensory nerve terminals of corneal cold receptors (Carr et al. 2003). During cooling, the shape of the ongoing positive–negative NTIs is more like that expected for an action potential invading the nerve termination electrotonically. Assuming that NTIs of this shape are produced by action potentials that propagate antidromically from their point of genesis into the nerve terminal (see above), the changes in NTI shape seen during cooling would be consistent with a depolarization-induced inactivation of Na+ channels reducing their availability to support active propagation in the nerve termination. Conversely, warming alters NTI shape in a manner consistent with an increased contribution of Na+ channels to action potentials propagating into the nerve terminal, an effect that can be accounted for by hyperpolarization of the nerve terminal reducing the number of voltage-inactivated Na+ channels. Here we report that in receptors where NTIs of both conventional and inverted (negative–positive) shape are recorded, heating increases the relative frequency of occurrence of inverted NTIs (Fig. 8). Furthermore, in approximately 10% of receptors for which only conventional shaped NTIs where recorded at ∼32°C, heating caused the appearance of inverted NTIs. As inverted NTIs represent initiation close to the nerve termination, these findings are also consistent with a hyperpolarization of the nerve terminal during warming increasing the number of Na+ channels available for activation in the nerve terminal axon and thereby increasing the likelihood that action potentials are initiated at sites located very close to the site of recording at the nerve termination.
The sensory nerve terminals of guinea-pig corneal cold-sensitive neurons possess both TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ channels (Brock et al. 1998, 2001; Carr et al. 2002). However, TTX-R Na+ channels alone are able to support the initiation of action potentials in cold receptors (Brock et al. 1998). In most cold receptors, under control conditions, blockade of both TTX-S and TTX-R Na+ channels selectively at the nerve terminal with the local anaesthetic, lignocaine, does not change the shape of positive–negative-shaped NTIs (Brock et al. 2001). However, hyperpolarization of the nerve terminal with extracellularly applied current reveals a large inward Na+ current that is abolished by lignocaine but is largely resistant to TTX (Carr et al. 2002). These findings demonstrate that, under control conditions, Na+ channels within the nerve terminal are substantially inactivated and that, when hyperpolarized, TTX-R Na+ channels make a dominant contribution to the Na+ current elicited when the action potential invades the nerve termination. Therefore, when warmed, the increased likelihood of action potentials being initiated at sites located very close to the nerve terminal is most probably due to relief of TTX-R Na+ channel inactivation.
In Trpm8GFP transgenic mice, axons expressing eGFP branch within the stromal nerve plexus but do not branch after entering the corneal epithelium within which they typically course for over 100 μm before terminating. Assuming a similar arrangement in the guinea-pig cornea, the length constant of the nerve terminal axons would need to be in the order of hundreds of micrometers if individual terminals of corneal cold receptors were to interact electrotonically. To estimate the length constant necessitates some assumptions regarding axon size as well as the specific resistivity of both the axoplasm and axolemma. In cornea, the axons terminating in the superficial layer of the corneal epithelium have diameters of 0.25 μm or less but the terminating axons are varicose (see Belmonte & Gallar, 1996). We have therefore assumed an average axon diameter of 0.5 μm. The specific resistivity of axoplasm has been determined in a variety of preparations and values in the range 50–100 Ω cm are consistently reported (Jack et al. 1975). Estimates of the specific resistivity of the membrane for mammalian sensory neurons vary considerably. For C-fibre neurones, values determined from somal recordings range from 2–7 kΩ cm2 (cat nodose ganglion: Gallego & Eyzaguirre, 1978; cat DRG: Brown et al. 1981) to 50 kΩ cm2 (rat DRG: Villiere & McLachlan, 1996) while for Aδ-fibre neurones values range between 2 kΩ cm2 (rat nodose ganglion: Gallego & Eyzaguirre, 1978) and 30 kΩ cm2 (rat DRG: Villiere & McLachlan, 1996). For specific membrane resistivities between 2–30 kΩ cm2, a specific axoplasmic resistivity of 100 Ω cm and an axonal diameter of 0.5 μm, the axonal length constant ranges between ca 220 and 860 μm. A length constant of this magnitude suggests that action potentials generating positive–negative-shaped NTIs may initiate at a site where sensory stimulus-induced changes in membrane potential generated in the nerve terminals of adjoining nerve terminal branches are integrated. This point may be proximal to the most distal axon branch and therefore within the stroma. However, it is also possible that action potential initiation occurs within the terminating axon branch as evidenced by the occurrence of negative–positive NTIs. Indeed, because we are only recording from a single nerve terminal, it is possible that action potentials are always initiated within a terminating axon and propagate by axon reflex to the site of recording generating a positive–negative-shaped NTI, in which case the low incidence of recording from nerve terminals with inverted shaped NTIs may simply reflect the likelihood of recording from the nerve terminal branch within which action potentials are being initiated. In either case, the estimate of axonal length constant relative to the morphology of cold-sensitive axons in the cornea suggests that sensory-induced changes in membrane potential from individual nerve terminal branches can interact within the nerve terminal arbour.
The recording of an action potential during its initiation provides evidence that the functional substrates responsible for sensory transduction and action potential initiation exist in parallel with one another within the nerve terminal region of unmyelinated sensory nerve terminals. This arrangement implies that in cold-sensitive nerve terminals, changes in membrane potential resulting from the transduction of temperature not only drive the initiation of action potentials but also affect directly the number of Na+ channels available for activation within the nerve terminal region. As a consequence impulse encoding during hyperpolarization is inextricably associated with a relief of Na+ inactivation and therefore a tendency for the site of action potential initiation to shift towards the nerve termination. Likewise during depolarization the likelihood of action potential initiation at sites more proximal to the axon termination increases.