T-type Ca2+ channels are required for enhanced sympathetic axon growth by TNFα reverse signalling

Tumour necrosis factor receptor 1 (TNFR1)-activated TNFα reverse signalling, in which membrane-integrated TNFα functions as a receptor for TNFR1, enhances axon growth from developing sympathetic neurons and plays a crucial role in establishing sympathetic innervation. Here, we have investigated the link between TNFα reverse signalling and axon growth in cultured sympathetic neurons. TNFR1-activated TNFα reverse signalling promotes Ca2+ influx, and highly selective T-type Ca2+ channel inhibitors, but not pharmacological inhibitors of L-type, N-type and P/Q-type Ca2+ channels, prevented enhanced axon growth. T-type Ca2+ channel-specific inhibitors eliminated Ca2+ spikes promoted by TNFα reverse signalling in axons and prevented enhanced axon growth when applied locally to axons, but not when applied to cell somata. Blocking action potential generation did not affect the effect of TNFα reverse signalling on axon growth, suggesting that propagated action potentials are not required for enhanced axon growth. TNFα reverse signalling enhanced protein kinase C (PKC) activation, and pharmacological inhibition of PKC prevented the axon growth response. These results suggest that TNFα reverse signalling promotes opening of T-type Ca2+ channels along sympathetic axons, which is required for enhanced axon growth.


Introduction
A variety of extracellular signals participate in regulating the establishment of sympathetic innervation in the developing peripheral nervous system [1,2]. The most extensively studied and best understood of these is nerve growth factor (NGF), a secreted protein synthesized in tissues innervated by NGFresponsive neurons. NGF promotes the survival of developing sympathetic neurons, and level of NGF synthesis in different tissues regulates the number of neurons that innervate these tissues by restricting the extent of cell death among the innervating neurons during development [3]. NGF also acts on sympathetic axon terminals within target tissues to promote growth and branching [4]. Whereas retrograde PI3-kinase/Akt signalling to the cell body plays an important role in mediating NGF-promoted survival, activation of ERK1/ ERK2 downstream of TrkA, the NGF receptor tyrosine kinase, plays a major role in mediating the local axon growth-promoting effects of NGF [5][6][7][8].
A recently discovered target-derived signal that also promotes sympathetic axon growth and branching during the stage in development when sympathetic axons are ramifying in their targets is tumour necrosis factor receptor 1 (TNFR1) [9]. TNFR1 expressed by sympathetic target tissues acts as a ligand for membrane-integrated TNFa expressed along sympathetic axons, and TNFR1-activated TNFa reverse signalling enhances sympathetic axon growth and branching. Mice lacking either TNFa or TNFR1 display greatly reduced sympathetic innervation density in multiple tissues, but unlike NGF-deficient mice, these mice show no deficits in sympathetic neuron number. As with NGF, activation of ERK1/ERK2 signalling plays a key role in mediating the axon growth-promoting effects of TNFa reverse signalling. Activation of ERK1/ERK2 by TNFa reverse signalling is due to rapid Ca 2þ influx [9]; however, the identity of the channels that open in response to TNFa reverse signalling is not known. Our aim here was to identify these channels, determine where on sympathetic neurons they are functionally relevant for axon growth in response to TNFR1-activated TNFa reverse signalling, and provide a link between Ca 2þ influx and ERK1/ERK2 activation. Using a combination of pharmacological studies, electrophysiology, Ca 2þ reporter studies and western blot analysis, we show that activation of TNFa reverse signalling in sympathetic axons increases T-type Ca 2þ channel activation and the subsequent activation of protein kinase C (PKC) and ERK1/ERK2 are required for enhanced axon growth.

T-type Ca 2þ channels are required for TNFR1-Fcpromoted axon growth
Our demonstration that activation of TNFa reverse signalling causes Ca 2þ influx in postnatal sympathetic neurons and that this is necessary for enhanced axon growth [9] implicates ligand or voltage-gated Ca 2þ channels in the axon growth response to TNFR1. To examine this, we first tested whether the broad-spectrum voltage-gated Ca 2þ channel blocker dotarizine [10,11] inhibits the growth response to TNFa reverse signalling. Low density dissociated cultures of superior cervical ganglion (SCG) neurons were plated in medium containing NGF to sustain their survival and were treated with a divalent TNFR1-Fc chimera to activate TNFa reverse signalling [9] in the presence and absence of dotarizine. After 24 h, quantification of the size and complexity of the neurite arbours showed that the TNFR1-Fc chimera caused highly significant increases in neurite length and branch point number (figure 1). Accordingly, the Sholl profiles, which plot neurite intersections with a series of concentric circles centred on the cell body, were clearly larger and more complex than those for neurons grown with NGF alone (control cultures). The size and complexity of the neurite arbours of neurons treated with dotarizine alone were not significantly different from those in control cultures. However, 1 mM dotarizine completely prevented TNFR1-Fc enhanced neurite growth (figure 1). These studies suggest that voltage-gated Ca 2þ channels at the plasma membrane are required for the axon growth enhancing effect of TNFa reverse signalling.
To ascertain which kinds of voltage-gated Ca 2þ channels are required for the enhanced axon growth response to TNFa reverse signalling, we carried out similar studies using subtype-selective Ca 2þ channel blockers [12]. For these experiments, we used 10 mM nifedipine which is a selective blocker of L-type Ca 2þ channels [13], 100 nM v-agatoxin TK which blocks P/Q-type Ca 2þ channels, [14], 10 nM v-grammotoxin SIA which blocks N-type and P/Q-type Ca 2þ channels [15,16], 60 nM SNX 482 which blocks R-type Ca 2þ channels [17] and several T-type Ca 2þ channel blockers, 1 mM mibefradil [18][19][20], 200 nM TTA-A2 [21] and 200 nM TTA-P2 [22]. None of these Ca 2þ channel inhibitors had any significant effect on the extent of NGF-promoted axon growth from P0 SCG neurons when added in the absence of TNFR1-Fc (figures 1 and 2). None of the blockers nifedipine, v-agatoxin TK, v-grammotoxin SIA or SNX 482 significantly affected TNFR1-Fc-enhanced axon growth (figure 1), suggesting that none of the L-type, N-type, P/Qtype or R-type Ca 2þ channels are required for the enhanced axon growth response to TNFR1-Fc. However, each of the T-type Ca 2þ channel blockers (mibefradil, TTA-A2 and TTA-P2) completely inhibited TNFR1-Fc-enhanced axon growth ( figure 2a,b). None of these T-type Ca 2þ channel blockers significantly affected neuronal survival either alone or in the presence of TNFR1-Fc at the concentrations used (not shown). These findings suggest that T-type Ca 2þ channels are required for TNFR1-enhanced axon growth.

Expression of T-type Ca 2þ channel mRNA in developing superior cervical ganglion neurons
TNFa reverse signalling enhances axon growth from SCG neurons during a narrow developmental window between P0 and P5 when sympathetic axons are ramifying extensively in their targets [9]. To confirm expression of transcripts encoding T-type Ca 2þ channels in SCG during this period of development, we used real-time PCR to quantify the levels of mRNAs encoding these channels in SCG dissected from mice at stages from E18 to P10. Separate genes, Ca v 3.1, Ca v 3.2 and Ca v 3.3, encode three T-type Ca 2þ channel isoforms that are all low voltage-activated and inactivating, but differ slightly in their biophysical properties and distribution [23]. Real-time PCR revealed that transcripts for all three genes are expressed throughout this period of development (figure 2c). There was an overall decrease in level of Ca v 3.1 mRNA with developmental age and the expression of Ca v 3.2 and Ca v 3.3 mRNAs peaked at P0 (figure 2c). These results are consistent with the expression of all three T-type Ca 2þ channel isoforms throughout the period of development when TNFa reverse signalling enhances axon growth.

T-type Ca 2þ currents are undetectable in superior cervical ganglion somata
Given our evidence for the involvement of T-type Ca 2þ channels in mediating the effects of TNFa reverse signalling on axon growth, we recorded Ca 2þ currents from SCG neuron somata using whole cell patch clamp ( figure 3). T-type channels are activated at low potentials (around -40 mV), and the current generated is characterized by a rapid activation and a rapid inactivation [23]. We initially recorded voltage-gated Ca 2þ currents in response to a test potential to 25 mV from a holding potential of 280 mV using 5 mM Ca 2þ as a charge carrier; in these conditions, T-type Ca 2þ channels should be maximally activated with a reduced contribution of high voltage-activated Ca 2þ current. Ca 2þ currents were recorded for 5 min before applying TNFR1-Fc and then recorded every 10 s for 10 -15 min (figure 3a). In control conditions, Ca 2þ currents exhibited only a sustained, non-inactivating, component characteristic of high rsob.royalsocietypublishing.org Open Biol. 7: 160288 rsob.royalsocietypublishing.org Open Biol. 7: 160288 voltage-activated Ca 2þ currents. After application of TNFR1-Fc, Ca 2þ currents were similar in shape and amplitude (figure 3a,b). In order to maximize the size of the currents, we raised the Ca 2þ concentration in the recording medium to 10 mM, and we compared the current -voltage relationships of the Ca 2þ currents before and after treatment with TNFR1-Fc (figure 3c,d). In these conditions, no low voltage-activated transient Ca 2þ current could be recorded after applying TNFR1-Fc and no difference in the current-voltage curves was recorded. Altogether, our results suggest that length (mm) Figure 3. Functional significance of T-type Ca 2þ channels in sympathetic axons. (a) Representative current traces resulting from a step potential from 280 mV to 25 mV before Fc fragment (control, black trace) and after 10 -15 min application of TNFR1-Fc (5 mg ml 21 ; red trace). Ca 2þ currents were recorded every 10 s before and after the application of TNFR1-Fc. Recordings were performed using 5 mM Ca 2þ as a charge carrier. No clear difference in calcium current shape and amplitude is visible between the control and the treatment with TNFR1 suggesting that no T-type currents are functionally expressed in the somata of SCG neurons.
(b) Bar chart shows the normalized peak Ca 2þ current amplitude recorded from SCG neurons before (filled bar) and 10 to 15 min after starting TNFR1-Fc treatment (open bar, 92 + 18%, n ¼ 7 cells, p ¼ 0.1328, paired t-test). (c) Representative current traces recorded from a TNFR1-treated SCG neuron in response to step potentials from 280 mV to between 260 mV to þ50 mV in 10 mV increments. Recordings were performed using 10 mM Ca 2þ as a charge carrier. rsob.royalsocietypublishing.org Open Biol. 7: 160288 TNFa reverse signalling does not induce the functional expression of T-type channels in the somata of SCG neurons.

T-type Ca 2þ channels are functionally relevant in sympathetic axons not in cell somata
The above results raise the possibility that T-type Ca 2þ channels are only functionally relevant for axon growth enhancement by TNFa reverse signalling along the axons themselves. To test this, we blocked these channels in a compartment culture paradigm in which the cell somata and growing axons are cultured in different compartments separated by a barrier (figure 3e). We previously reported that addition of TNFR1-Fc to the axon compartment, but not to the soma compartment, enhances axon growth [9]. Here we studied the consequences of blocking T-type Ca 2þ channels separately in the soma and axon compartments on TNFR1-Fc-enhanced axon growth. In these experiments, we seeded P0 SCG neurons into one compartment (the soma compartment) of a two-compartment device that contained NGF in both compartments to sustain neuronal survival and encourage axon growth from the soma compartment into the axon compartment. After a 24 h incubation, we labelled the axons in the axon compartment with the fluorescent vital dye calcein-AM, which also retrogradely labelled cell bodies of neurons that projected axons into the axon compartment.
We used a stereological method to quantify the extent of axon growth in the axon compartment relative to the number of neurons projecting axons into this compartment. Addition of the T-type Ca 2þ channel blocker TTA-P2 to either the axon compartment or the soma compartment had no significant effect on the extent of axon growth in the axon compartment in cultures that were not treated with TNFR1-Fc (figure 3f,g). Addition of TNFR1-Fc to the axon compartment significantly enhanced axon growth in this compartment. Enhanced axon growth was completely inhibited by the addition of TTA-P2 to the axon compartment, but not by TTA-P2 addition to the soma compartment (figure 3f,g). Similar experiments using the T-type Ca 2þ channel blocker mibefradil yielded very similar results (not shown). These findings suggest that T-type Ca 2þ channels are functionally relevant for axon growth enhancement by TNFa reverse signalling in axons, but not in somata.

TNFa reverse signalling induces Ca 2þ transients in sympathetic axons via T-type Ca 2þ channels
To determine whether activation of TNFa reverse signalling induces Ca 2þ transients in sympathetic axons, P0 SCG neurons were co-transfected with plasmids expressing mCherry or DsRed (to identify transfected neurons) and a genetically encoded Ca 2þ sensor (GCaMP6s-CAAX) whose expression is targeted to the plasma membrane [24]. After transfection, the neurons were cultured for 12 h in medium containing 10 ng ml 21 NGF and 300 nM TAPI-O (TNFa processing inhibitor, which inhibits TACE and thereby maintains the level of membrane-integrated TNFa at the plasma membrane). Variations in fluorescence were then recorded in processes, and growth cones of SCG neurons perfused with a saline solution for 5 min before adding Fc fragment control protein or TNFR1-Fc (figure 4a). Prior to treatment with these reagents, there was a low frequency of Ca 2þ transients. When analysed at 5 min intervals after the start of treatment, the signal frequency became significantly greater in TNFR1-Fc-treated cultures compared with Fc-treated control cultures between 5 and 10 min after the start of treatment, and remained significantly greater at each subsequent 5 min interval until the end of the recording period at 25 min (figure 4b).
Interestingly, Ca 2þ transients were local events and did not propagate to the rest of the neuron. These results suggest that TNFR1-Fc increases the frequency of Ca 2þ transients. Integration of the areas under individual fluorescent peaks above a standard threshold revealed significant increases in TNFR1-Fc-treated cultures compared with Fc-treated cultures during each 5 min interval, starting at the 5-10 min interval (figure 4c). However, there was no significant increase in the amplitude of individual Ca 2þ transient peaks in TNFR1-Fc-treated cultures compared with Fc-treated controls (figure 4d). Taken together, these results suggest that TNFR1-Fc not only increases the frequency of Ca 2þ signals but also increases the duration of individual events. These significant increases in both the frequency and duration of fluorescent signals brought about by TNFR1-Fc were completely prevented by TTA-P2 (figure 4b,c), suggesting that these events were mediated by T-type Ca 2þ channels. In addition to the temporal characteristics of fluorescence signals, differences were observed in their spatial distribution.

TNFR1-Fc-enhanced axon growth occurs independently of action potential generation
Because activation of T-type Ca 2þ channels has been shown to trigger a burst of action potentials mediated by Na þ channels in many different kinds of neuron [23], we investigated whether action potential generation plays a role in TNFRl-Fc-promoted axon growth. To test this, we treated P0 SCG neurons with tetrodotoxin (TTX), a selective blocker of most voltage-gated Na þ channels and inhibitor of action potential generation in neurons. TTX did not inhibit TNFR1-Fcpromoted neurite growth, suggesting that the generation of action potentials is not required for enhanced axon growth (figure 5a). T-type channels are known to participate in low threshold oscillations in other tissues [25].

Protein kinase C activation is required for TNFR1-Fc-promoted axon growth
We have previously shown that TNFa reverse signalling enhances axon growth by activating MEK/ERK of the MAP kinase signalling cascade [9]. To elucidate the link between T-type Ca 2þ channels, Ca 2þ influx, elevation of [Ca 2þ ] i and MEK/ERK activation, we explored the potential role of PKC, a family of serine -threonine protein kinases that has been implicated in activating multiple signalling cascades, including MAP kinase signalling [26 -28]. Of the 10 known PKC members, the conventional isozymes (PKC-a, PKC-bI, PKC-bII and PKC-g) are activated by both [Ca 2þ ] i and diacylglycerol [29]. We began our investigation of the potential role of PKC in TNFR1-Fc-promoted axon growth by ascertaining whether TNFR1-Fc activates PKC in cultured SCG neurons.
In these experiments, we first cultured P0 SCG neurons for 12 h with NGF before treating them with TNFR1-Fc. Western blot analysis revealed an increase in the level of phosphoserine 660 PKC-bII after 15 min exposure to TNFR1-Fc. This rsob.royalsocietypublishing.org Open Biol. 7: 160288 increase was prevented by preincubating the neurons with mibefradil (figure 5b).
To determine whether PKC activation is required for ERK1/ERK2 activation by TNFR1-Fc, we studied the effect of GF 109203X, a potent and selective inhibitor of PKC [30].
In these experiments, we first cultured P0 SCG neurons for 12 h with NGF before treating them with TNFR1-Fc. Western blot analysis revealed that the increases in phospho-ERK1 and phospho-ERK2 brought about by TNFR1-Fc treatment were partially or fully prevented by preincubating the  rsob.royalsocietypublishing.org Open Biol. 7: 160288 neurons with GF 109203X, and that GF 109203X alone did not significantly affect the levels of phospho-ERK1 and phospho-ERK2 (figure 5c). Finally, to investigate if PKC activation is required for TNFR1-Fc-promoted neurite growth, we examined whether GF 109203X could prevent this. In these experiments, we plated P0 SCG neurons in NGF-containing medium, pretreated them for 2 h with GF 109203X before adding TNFR1-Fc and imaging the neurite arbours 24 h later. GF 109203X prevented TNFR1-Fc enhanced neurite growth, as shown by quantification of neurite length, branch point number and Sholl analysis (figure 5d). Taken together, these findings suggest that PKC plays a role in mediating the effect of TNFa reverse signalling on axon growth.

Discussion
The data reported here establish the essential role of T-type Ca 2þ channels in mediating the effects of TNFa reverse signalling on axon growth in developing sympathetic neurons. We show that each of three selective T-type Ca 2þ channel inhibitors, mibefradil, TTA-A2 and TTA-P2, completely prevent TNFR1-Fc-promoted axon growth without affecting axon growth in the absence of TNFR1-Fc or affecting neuronal survival. In contrast, blockers of L-type, N-type, P/Q-type and R-type Ca 2þ channels have no effect on TNFR1-Fc-promoted axon growth. Furthermore, we show that TNFR1-Fc, but not Fc control protein, significantly increases the frequency and duration of Ca 2þ transients recorded using a genetically encoded Ca 2þ sensor targeted to the plasma membrane, and that pharmacological blockade of T-type Ca 2þ channels completely prevents these increases. Given the importance of Ca 2þ influx and subsequent elevation of intracellular Ca 2þ to TNFR1-Fc-promoted axon growth [9], our findings show that Ca 2þ influx triggered by activation of TNFa reverse signalling is mediated by T-type Ca 2þ channels and further support the crucial role of T-type Ca 2þ channels in TNFR1-Fc-promoted axon growth.
Our compartment culture results suggest that T-type channels are only functionally relevant in axons for TNFR1promoted growth. TNFR1 only promoted axon growth when applied to axons not somata and T-type Ca 2þ channel inhibitors only inhibited TNFR1-promoted axon growth when applied to axons not somata. These results are consistent with our whole-cell patch-clamp studies which did not detect T-type Ca 2þ currents at the somata. The absence of T-type Ca 2þ currents in the somata of sympathetic neurons, as also shown in rat and frog SCG neurons [31 -33], implies that T-type Ca 2þ channels are not expressed in this location in sufficient numbers to be detected. T-type calcium channels have only been identified in the cell bodies of a small subset of sympathetic neurons [33]. This could be because these Ca 2þ channels migrate into axons as the neurons extend axons in culture or because Ca 2þ channels are synthesized locally in axons because the encoding mRNA is transported along axons prior to translation [34]. However, they were not previously detected in developing axons or growth cones of frog superior cervical neurons [33]. We detected transcripts encoding all three T-type Ca 2þ channel isoforms in SCG throughout the period of development when TNFa reverse signalling enhances axon growth. This raises the possibility that all three subtypes could contribute to axon growth, in the presence of TNFR1. Because subtype-specific T-type Ca 2þ channel inhibitors are not currently available, we were unable to determine pharmacologically whether a particular subtype plays a more prominent role than others. While various anti-T-type channel antibodies have been reported, they have questionable specificity for different T-type Ca 2þ channel isoforms and are thus of limited use for immunocytochemical studies of the distribution of these channel proteins.
Our imaging studies show that TNFa reverse signalling modulates Ca 2þ signalling by affecting the frequency and duration of discrete events. TNFR1-Fc causes highly significant increases in the frequency and duration of intracellular Ca 2þ transients and these increases are prevented from occurring by T-type Ca 2þ channel blockers. However, the T-type channel blocker did not abolish these events under control conditions, indicating that other Ca 2þ channels are also involved in these Ca 2þ transients, and opening the possibility that T-type channels were either inserted in the membrane by TNFR1-Fc, or it caused a shift in the window current for T-type channels [23], resulting in their increased availability at the resting membrane potential in the axons.
An interesting issue is how TNFa reverse signalling influences the opening of T-type channels. It is well documented that T-type Ca 2þ channels open secondary to small membrane depolarizations [23,35]. Thus, it is possible that TNFa reverse signalling causes T-type Ca 2þ channel opening by first causing membrane depolarization. However, our current clamp studies did not provide any evidence in support of this in the cell body (data not shown). This raises the possibility that TNFa reverse signalling only affects the membrane potential in the developing sympathetic axons, inducing oscillations, or affects T-type channels by another means, for example by changing the voltage-dependence of the window current, which is maximal at about 260 mV in 2 mM Ca 2þ [36]. There is evidence of numerous post-translational modifications of channels that affect T-type channel properties [37,38].
In this study, we have demonstrated the crucial role of T-type Ca 2þ channels in mediating the effects of TNFa reverse signalling on sympathetic axon growth. TNFa reverse signalling has also been reported to enhance the growth of sensory axons [39], which raises the question of whether T-type Ca 2þ channels play a role in sensory axon growth. In addition to the effects of TNFa reverse signalling on axon growth, an additional example of reverse signalling within the TNF superfamily affecting axon growth has recently been reported. CD40 ligand (CD40L, TNFSF5) reverse signalling in subsets of sympathetic neurons enhances axon growth, and this is required in vivo for the ramification of axons in the tissues innervated by these neurons [40]. Again, it would be of interest to determine whether T-type Ca 2þ channels and Ca 2þ influx play any role in mediating CD40 L reverse signalling in developing sensory neurons, where T-type channels have been identified in the somata of specific subtypes [41,42], and play important physiological and pathological roles [43].
We have previously shown that ERK1/ERK2 is activated in developing sympathetic neurons by TNFR1-Fc and that pharmacological inhibition of ERK1/ERK2 activation prevents the axon growth response to TNFR1-Fc [9]. To ascertain the link between TNFR1-Fc-promoted T-type Ca 2þ channel opening and ERK activation, we explored the rsob.royalsocietypublishing.org Open Biol. 7: 160288 potential role of PKC, which is activated by Ca 2þ signals and activates in turn a variety of signalling pathways including ERK [29]. We showed that TNFR1-Fc enhanced PKC activation and that pharmacological inhibition of PKC reduced ERK1/ERK2 activation by TNFR1-Fc. Importantly, pharmacological inhibition of PKC completely prevented the axon growth response of sympathetic neurons to TNFR1-Fc. Taken together, our findings have established a sequence of essential steps that link TNFa reverse signalling with enhanced axon growth in developing sympathetic neurons. Activation of TNFa reverse signalling enhances intracellular Ca 2þ transients mediated by T-type Ca 2þ channels in axons, leading to activation of PKC and activation of ERK1/ERK2. In future work, it will be particularly interesting to establish how TNFa reverse signalling modulates the opening of T-type Ca 2þ channels, initiating this chain of events.

Conclusion
We have discovered a novel and unexpected function for voltage-gated T-type Ca 2þ channels in axons of SCG sympathetic neurons, which do not exhibit T-type channels in their somata. We show that these channels mediate Ca 2þ transients in developing sympathetic axons following activation of TNF reverse signalling and that they are essential for the enhanced axon growth and branching promoted by TNF reverse signalling. Given the physiological significance of TNF reverse signalling for establishing sympathetic innervation, this constitutes a clear role for voltage-gated T-type Ca 2þ channels in sympathetic neuron development.

Neuron culture
Dissected SCG were freed of adherent connective tissue using tungsten needles and were trypsinized and plated at very low density (approx. 200 neurons per dish/well) in polyornithine and laminin-coated 35 mm tissue culture dishes (Greiner, Gloucestershire, UK) or four-well dishes (Starlab, Milton Keynes, UK) in serum-free Hams F14 medium [44] supplemented with 0.25% Albumax I (Invitrogen, Paisley, UK). Neuronal survival was estimated by counting the number of neurons in four-well dishes 2 h after plating and again at 24 h. All neurons in each well were counted. The number of neurons surviving at 24 h was expressed as a percentage of the initial number of neurons counted. Analysis of the size and complexity of neurite arbours was carried out in 35 mm dishes 24 h after plating. The neurite arbours were labelled by incubating the neurons with the fluorescent vital dye calcein-AM (1 : 1000, Invitrogen, Paisley, UK) at the end of the experiment. Images of neurite arbours were acquired by fluorescence microscopy and analysed to obtain total neurite length, number of branch points and Sholl profiles [45].
For studying the effects of regional blockade of T-type Ca 2þ channels on neurite growth, P0 SCG neurons were plated in one compartment of a two-compartment microfluidic device (Xona microfludics, CA, USA). Both compartments received NGF and the TNFR1-Fc was added to the axon compartment. A T-type Ca 2þ channel blocker was added to either the soma or axon compartment. After 24 h incubation, the axons in the axon compartment and the cell bodies that projected axons into this compartment were labelled by adding the fluorescent vital dye calcein-AM to the axon compartment. Axon length was quantified by a modification of a previously described method [46]. Briefly, using NIH IMAGEJ, a grid of vertical lines was traced with an interline interval of 200 mm. Total intersections between neurites and the grid were counted and normalized against the number of labelled somas in the cell body compartment. Average neurite length per projecting cell body was calculated using the formula L ¼ pDI/2, where L is the estimated length, D is the interline interval and I the average number of intersections per projecting cell body. Measurements were independently carried out in all fields along the microfluidic barrier.

GCaMP imaging in superior cervical ganglion neurons
SCG neurons were co-transfected with either 0.8 mg pCAGGs-mCherry (Addgene) or 0.8 mg pDsRed-Express-N1 (Clontech) and 2.5 mg membrane-directed pGPCMV-GCaMP6s-CAAX (Addgene) using a microporator (Digital Bio; 2 Â 30 ms pulse at 900 V) and cultured at a high density in medium containing 10 ng ml 21  Measurement and analysis were performed as previously described [47]. Normalized current -voltage relationships were fitted with a modified Boltzmann equation as follows: I ¼ G max Â (V 2 V rev )/(1 þ exp( 2 (V 2 V 50,act )/k)), where I is the current density (in picoamperes Â picofarad 21 ), G max is the maximum conductance (in nanosiemens Â picofarad 21 ), V rev is the reversal potential in mV, V 50,act is the midpoint voltage for current activation in mV, and k is the slope factor.

Real-time QPCR
The levels of Cav3.1, Cav3.2 and Cav3.3 mRNAs were quantified by RT-QPCR relative to a geometric mean of mRNAs for the housekeeping enzymes glyceraldehyde phosphate dehydrogenase (GAPDH), succinate dehydrogenase (SDHA) and hypoxanthine phosphoribosyltransferase 1 (HPRT1). Total RNA was extracted from whole SCG with the RNeasy Mini extraction kit (Qiagen, Crawely, UK), and 5 ml was reverse transcribed for 1 h at 458C using the Affinity-Script kit (Agilent, Berkshire, UK) in a 25 ml reaction according to the manufacturer's instructions. cDNA (2 ml) was amplified in a 20 ml reaction volume using Brilliant III ultrafast QPCR master mix reagents (Agilent, Berkshire, UK). QPCR products were detected using dual-labelled (FAM/BHQ1) hybridization probes specific to each of the cDNAs (MWG/Eurofins, Ebersberg, Germany). The PCR primers were: gapdh forward Forward and reverse primers were used at a concentration of 150 nM each and dual-labelled probes were used at a concentration of 300 nM. PCR was performed using the Mx3000P platform (Agilent, Berkshire, UK) using the following conditions: 45 cycles of 958C for 12 s and 608C for 35 s. Standard curves were generated in every 96-well plate, for each cDNA for every real-time PCR run, by using serial threefold dilutions of reverse transcribed spleen total RNA (Ambion, Paisley, UK). Three separate dissections were performed for each age.

Statistical analysis
Statistical comparisons were performed by independent Student's t-test or one-way ANOVA followed by Fisher's post hoc test.
Ethics. Breeding and housing of mice (Mus musculus) was approved by the Cardiff University Ethical Review Board and was performed within the guidelines of the Home Office Animals (Scientific Procedures) Act, 1986.