Functional tetrodotoxin-resistant Na+ channels are expressed presynaptically in rat dorsal root ganglia neurons

Neuroscience 159 (2009) 559 –569

FUNCTIONAL TETRODOTOXIN-RESISTANT Naⴙ CHANNELS ARE EXPRESSED PRESYNAPTICALLY IN RAT DORSAL ROOT GANGLIA NEURONS Y. V. MEDVEDEVA, M.-S. KIM, K. SCHNIZLER AND Y. M. USACHEV*

channels (Kostyuk et al., 1981). Two TTX-R channels, Nav1.8 and Nav1.9 (Akopian et al., 1996; Dib-Hajj et al., 1998; Tate et al., 1998), are thought to be particularly important for the development of inflammatory, and possibly, neuropathic pain. Both channels are found primarily in nociceptive C- and A␦-fibers, and their expression and distribution are altered in response to inflammation or nerve injury (Cummins and Waxman, 1997; Novakovic et al., 1998; Tate et al., 1998; Amaya et al., 2000; Fang et al., 2002; Djouhri et al., 2003; Gold et al., 2003). Deletion of the Nav1.8 or Nav1.9 genes or the pharmacological inhibition of Nav1.8, markedly reduces inflammatory pain (Akopian et al., 1999; Priest et al., 2005; Amaya et al., 2006; Ekberg et al., 2006; Jarvis et al., 2007). There is also some evidence that Nav1.8, but not Nav1.9, contributes to neuropathic pain (Lai et al., 2002; Roza et al., 2003; Priest et al., 2005; Amaya et al., 2006; Jarvis et al., 2007). TTX-R Na⫹ channels are found on the cell body of sensory neurons in dorsal root ganglia (DRG) and in the peripheral and central processes of primary afferent neurons (Novakovic et al., 1998; Amaya et al., 2000). The density of the channels seems to be especially high in unmyelinated C-fibers, where the channels support propagation of action potentials and sensory synaptic transmission even in the presence of tetrodotoxin (TTX) (Yoshimura and Jessell, 1990; Jeftinija, 1994; Quasthoff et al., 1995; Gu and Macdermott, 1997) [but see; Pinto et al., 2008]. Furthermore, intraspinal administration of selective Nav1.8 inhibitors attenuates the activity of spinal neurons in neuropathic rats (McGaraughty et al., 2008) and reduces inflammatory and neuropathic pain sensitization (Ekberg et al., 2006). Despite the potential importance of TTX-R Na⫹ channels for sensory synaptic transmission and spinal pain processing, little is known about presynaptic expression of TTX-R Na⫹ channels. Interestingly, VGSCs are excluded from presynaptic terminals in some synapses, such as neuromuscular junction and the calyx of Held (Brigant and Mallart, 1982; Leao et al., 2005). On the contrary, VGSCs are found both in presynaptic boutons and on parent axons of mossy fibers in the hippocampus (Engel and Jonas, 2005). While axonal VGSCs control the speed of the action potential propagation, presynaptic VGCSs seem to be particularly important for regulating the action potential amplitude and the extent of presynaptic Ca2⫹ influx that triggers transmitter release (Engel and Jonas, 2005). TTX-R VGSCs play a crucial role in shaping action potentials in sensory neurons and are highly sensitive to prostaglandin E2 and other pain mediators (Gold et al., 1996; Renganathan et al., 2001; Bhave and Gereau,

Department of Pharmacology, University of Iowa, Carver College of Medicine, 2-250 BSB, 51 Newton Road, Iowa City, IA 52242, USA

Abstract—The tetrodotoxin-resistant (TTX-R) voltage-gated Naⴙ channels Nav1.8 and Nav1.9 are expressed by a subset of primary sensory neurons and have been implicated in various pain states. Although recent studies suggest involvement of TTX-R Naⴙ channels in sensory synaptic transmission and spinal pain processing, it remains unknown whether TTX-R Naⴙ channels are expressed and function presynaptically. We examined expression of TTX-R channels at sensory synapses formed between rat dorsal root ganglion (DRG) and spinal cord (SC) neurons in a DRG/SC co-culture system. Immunostaining showed extensive labeling of presynaptic axonal boutons with Nav1.8- and Nav1.9-specific antibodies. Measurements using the fluorescent Naⴙ indicator SBFI demonstrated action potential–induced presynaptic Naⴙ entry that was resistant to tetrodotoxin (TTX) but was blocked by lidocaine. Furthermore, presynaptic [Ca2ⴙ]i elevation in response to a single action potential was not affected by TTX in TTX-resistant DRG neurons. Finally, glutamatergic synaptic transmission was not inhibited by TTX in more than 50% of synaptic pairs examined; subsequent treatment with lidocaine completely blocked these TTX-resistant excitatory postsynaptic currents. Taken together, these results provide evidence for presynaptic expression of functional TTX-R Naⴙ channels that may be important for shaping presynaptic action potentials and regulating transmitter release at the first sensory synapse. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: Nav1.8, Nav1.9, SBFI, tetrodotoxin, synaptic transmission, sensory synapse.

Voltage-gated sodium channels (VGSCs) control the generation and propagation of action potentials. Several types of VGSCs are expressed in sensory neurons including Nav1.1, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 (Lai et al., 2004; Wood et al., 2004; Cummins et al., 2007). Pharmacologically, the VGSCs are subdivided into tetrodotoxinsensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na⫹ *Corresponding author. Tel: ⫹1-319-335-9388; fax: ⫹1-319-335-8930. E-mail address: [email protected] (Y. M. Usachev). Abbreviations: ara-C, cytosine-A-D-arabinofuranoside; [Ca2⫹]i, intracellular Ca2⫹ concentration; DRG, dorsal root ganglia; EGFP, enhanced green fluorescent protein; EPSC, excitatory postsynaptic current; FIV, feline immunodeficiency virus; HH buffer, Hepes-buffered recording solution; [Na⫹]i, intracellular Na⫹ concentration; P, postnatal day; PSD95, postsynaptic density 95 protein; SC, spinal cord; TTX, tetrodotoxin; TTX-R, tetrodotoxin-resistant; TTX-S, tetrodotoxin-sensitive; VGSC, voltage-gated sodium channel. 0306-4522/09 © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.12.029

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2004; Rush and Waxman, 2004). Thus, it is important to know whether or not TTX-R Na⫹ channels are expressed and function presynaptically. Here, we used the DRG/spinal cord (SC) co-culture system (Medvedeva et al., 2008) to examine presynaptic expression of functional TTX-R Na⫹ channels at the first sensory synapse under highly defined recording conditions. Fluorescent imaging demonstrated that action potential–induced presynaptic influx of Na⫹ and Ca2⫹ was resistant to TTX in a subset of DRG neurons. We also found that evoked glutamatergic synaptic transmission persisted in the presence of TTX in a large proportion of synaptic pairs examined. Furthermore, immunostaining revealed that both TTX-R Na⫹ channels, Nav1.8 and Nav1.9, were co-localized with the presynaptic proteins synaptophysin and bassoon in presynaptic boutons of DRG neurons. Taken together, these data suggest that functional TTX-R Na⫹ channels are expressed presynaptically in sensory neurons.

EXPERIMENTAL PROCEDURES Cell culture DRG/SC co-cultures were prepared as previously described (Medvedeva et al., 2008). In brief, newborn (postnatal days (P)0 –2) Sprague–Dawley rats were sacrificed according to a protocol approved by the University of Iowa Institutional Animal Care and Use Committee. All experimental procedures were approved by the University of Iowa Institutional Animal Care and Use Committee and were in accordance with the guidelines of the National Institutes of Health. Every effort was made to minimize the number of animals used and their suffering. DRGs were dissected from the thoracic and lumbar segments and incubated in pronase E dissolved in DMEM (1 mg/ml) for 7 min at 37 °C in a 10% CO2 incubator. Ganglia were washed twice in cold DMEM containing Hepes (20 mM; pH⫽7.4) and then mechanically dissociated by trituration with flame-constricted Pasteur pipettes of decreasing diameter. The SC was dissected into small segments (⬃0.5 mm) and then digested in trypsin solution (0.025% in DMEM) for 8 min at 37 °C in a 10% CO2 incubator. SC segments were then washed and dissociated using the procedure described above for DRG dissociation. Suspensions of SC and DRG cells were plated onto 25 mm glass coverslips coated with poly-L-ornithine and laminin. Cells were grown in DMEM medium supplemented with 5% heatinactivated horse serum, 5% fetal bovine serum and penicillin– streptomycin (100 U/ml and 100 ␮g/ml, respectively) in a 10% CO2 incubator at 37 °C. After 48 h, 5 ␮M cytosine-A-D-arabinofuranoside (ara-C) was added to cultures for 24 h. Two days later, cells were again treated with 5 ␮M ara-C for 24 h. Cultures were grown for 10 –16 days before use; 50% of the culture medium was replaced every 5– 6 days.

Lentivirus (feline immunodeficiency virus (FIV))Mediated Gene Transfer to Neurons pSynaptophysin-EGFPN1 was a gift from Dr. Jane Sullivan (University of Washington, Seattle, WA, USA). The entire synaptophysin– enhanced green fluorescent protein (EGFP) coding sequence was PCR-amplified (forward primer: 5=-GCTCTAGAGCCACCATGGACGTGGTG-3=; reverse primer: 5=-ACGCGTCGACCCGCTTTACTTGTACAGCTCG-3=), cut with XbaI and SalI and inserted into the FIV vector plasmid pVETL-Cmcs (Johnston et al., 1999). Synaptophysin-EGFP-containing FIV vectors were prepared by the University of Iowa Gene Transfer Vector Core, as previously described (Johnston et al., 1999). After 5– 6 days in

culture, DRG/SC co-cultures were incubated with the corresponding FIV vectors (0.5–1⫻104 pfu/ml) for 4 h. Cells were used within 4 – 8 days after infection. Transfection efficiency ranged from 10% to 30%.

Immunocytochemistry DRG/SC co-culture immunostaining was performed as previously described (Usachev et al., 2002; Medvedeva et al., 2008). The following primary antibodies were used: monoclonal mouse antiBassoon (1:500; StressGen/Assay Designs, Ann Arbor, MI, USA), monoclonal mouse anti-synaptophysin (0.5 ␮g/ml; Boehringer Mannheim), monoclonal mouse anti–postsynaptic density 95 (PSD95) (1:800; NeuroMab, Davis, CA, USA), polyclonal rabbit anti-Nav1.8 (1:1000 or 1:4000; SNS11; gift from Dr. John Wood, University College London; Djouhri et al., 2003) and polyclonal rabbit anti-Nav1.9 (1:1000 or 1:4000; Sigma, St. Louis, MO, USA). Alexa488-labeled goat antimouse serum and Alexa555-labeled goat antirabbit serum (1:200; Invitrogen/Molecular Probes, Eugene, OR, USA) were used as secondary antibodies. Alexa488 (␭ex⫽488 nm; ␭em⫽515 (10) nm) and Alexa555 (␭ex⫽543 nm; ␭em⫽580 (20) nm) fluorescence was observed using an Olympus BX61 microscope equipped with the Fluoview 300 laser-scanning confocal imaging system and a 60⫻ oil-immersion objective (NA⫽1.40).

Electrophysiology Whole-cell patch-clamp recordings were obtained using a patchclamp amplifier Axopatch 200 B, and an analog-to-digital converter Digidata 1322 A (Molecular Devices, Union City, CA), as previously described (Medvedeva et al., 2008). Data were collected (filtered at 5 kHz and sampled at 10 kHz) and analyzed using the pCLAMP 9 software (Molecular Devices, Union City, CA, USA). Patch pipettes were pulled from borosilicate glass (Narishige; 3–5 M⍀) on a Sutter Instruments P-87 micropipette puller and filled with the following solution (mM): 125 Kgluconate, 10 KCl, 3 Mg-ATP, 1 MgCl2, 5 EGTA, 10 Hepes, pH 7.25 adjusted with KOH (290 mOsm/kg with sucrose). The standard extracellular Hepesbuffered recording solution (HH buffer) contained (mM): 140 NaCl, 5 KCl, 1.3 CaCl2, 0.4 MgSO4, 0.5 MgCl2, 0.4 KH2PO4, 0.6 Na2HPO4, 3 NaHCO3, 10 glucose, 10 Hepes, pH 7.35 with NaOH (310 mOsm/kg with sucrose). Action potentials were evoked in the current-clamp mode by injecting 4 ms current pulses of incremental size (50 pA increment). Evoked excitatory postsynaptic currents (EPSCs) were recorded from SC neurons voltage-clamped at ⫺60 mV in the presence of the selective GABAA receptor antagonist bicuculline (10 ␮M) and the glycine receptor antagonist strychnine (2 ␮M). EPSCs were elicited every 4 s using a glass extracellular stimulating electrode (0.2– 0.4 ms pulse in control conditions and 0.4 – 0.8 ms in the presence of 200 or 500 nM TTX) positioned near the axon of a nearby DRG neuron (30 –50 ␮m away from the cell body). Stimuli were generated by a Grass S-44 stimulator and a stimulus isolation unit (Quincy, MA, USA). EPSC amplitude and latency were obtained for each synaptic pair/condition by averaging data from 20 to 30 EPSC traces. Data are presented as mean⫾SEM, with n representing the number of synaptic pairs tested.

Intracellular Ca2ⴙ concentration ([Ca2ⴙ]i) measurements in presynaptic boutons of DRG neurons Presynaptic [Ca2⫹]i measurements in DRG neurons have been previously described in detail (Medvedeva et al., 2008). In brief, small- to medium-size DRG neurons (⬍28 ␮m diameter) were loaded with bis-fura via patch pipettes by holding neurons in the whole-cell configuration for 5–7 min; the pipette was withdrawn

Y. V. Medvedeva et al. / Neuroscience 159 (2009) 559 –569 and recordings began within 20 –30 min. Only DRG neurons with resting membrane potential more negative than ⫺50 mV were used. The pipette solution contained (mM): 125 Kgluconate, 10 KCl, 3 Mg-ATP, 1 MgCl2, 10 Hepes, 0.2 bis-fura, pH 7.25 adjusted with KOH (290 mOsm/kg with sucrose). During the time of bis-fura loading, neurons were tested for their ability to generate all-ornone action potentials in the presence of 200 nM (500 nM in some experiments) TTX (Fig. 2A, insets), which is consistent with the TTX concentrations commonly used to block TTX-S Na⫹ channels (Gold et al., 1996; Cummins and Waxman, 1997; Renganathan et al., 2001; Jin and Gereau, 2006). At this concentration TTX completely blocked voltage-gated Na⫹ currents in SC neurons (n⫽6) and hippocampal neurons (n⫽8) that do not express TTX-R channels. In some experiments cells were transfected with synaptophysin-EGFP to facilitate visualization of presynaptic boutons. All measurements were performed using a flow-through chamber mounted on the stage of an inverted epifluorescent IX-71 microscope (Olympus, Japan). Fluorescence was alternately excited at 340 (12 nm bandpass) and 380 (12) nm using the polychrome IV monochromator (TILL Photonics, Germany). Excitation light was reflected off a dichroic mirror (410 nm, TILL Photonics) and focused on the cells via a 40⫻ oil-immersion objective (NA⫽1.35, Olympus). Emitted fluorescence was collected at 510 (84) nm (single band filter; Semrock, Rochester, NY, USA) using an IMAGO CCD camera (TILL Photonics). Pairs of 340/380 nm images were sampled at 10 Hz (Fig. 4A) or at 12.5 Hz (Fig. 4B–D). Fluorescence was corrected for background, as determined prior to the loading of cells with the indicator. Propagating action potentials were elicited in DRG neurons by using a glass extracellular stimulating electrode (0.2– 0.4 ms pulse in control conditions and 0.4 – 0.8 ms in the presence of 200 nM TTX) positioned near the cell body, as described above for the EPSCs recordings. In some experiments (Fig. 4B, C), presynaptic [Ca2⫹]i transients were studied using the whole-cell current-clamp configuration. In this case, action potentials were evoked under control conditions or in the presence of 500 nM TTX by injecting depolarizing current (2 ms pulses, 200 – 400 pA). Localized [Ca2⫹]i elevations within a small (1–3 ␮m) axonal region were elicited by focal stimulation via a saline-filled glass microelectrode (resistance 5– 8 M⍀, 3– 6 ms pulses) positioned very close (2–5 ␮m) to the axon. This approach is similar to the technique employed by another group to stimulate local Ca2⫹ influx into single presynaptic GABAergic boutons (Kirischuk et al., 1999). In order to block the spread of action potentials during focal stimulation, we added 1 ␮M TTX and lowered the extracellular Na⫹ concentration to 35 mM by replacing 105 mM NaCl with choline-Cl in the extracellular solution. Under these conditions, action potentials are completely blocked in both TTX-sensitive and TTX-resistant DRG neurons (Medvedeva et al., 2008).

Intracellular Naⴙ concentration ([Naⴙ]i) measurements in presynaptic boutons of DRG neurons All experiments were performed on small-to medium-size DRG neurons (⬍28 ␮m diameter) using the current-clamp mode of patch clamp. Only DRG neurons with resting membrane potential more negative than ⫺50 mV were used. The Na⫹ indicator SBFI was included in the following pipette solution (mM): 125 Kgluconate, 10 KCl, 3 Mg-ATP, 1 MgCl2, 10 Hepes, 1 SBFI, 0.1 EGTA, pH 7.25 adjusted with KOH (290 mOsm/kg with sucrose). [Na⫹]i measurements were performed using either single- or dual-wavelength fluorescence mode. For the single-wavelength recordings (Fig. 2), SBFI fluorescence was excited at 340 (12) and collected at 510 (84) nm, at a sampling frequency of 3 Hz. [Na⫹]i changes were presented as ⌬F/F0⫽(F–F0)/F0, where F is current fluorescence intensity, and F0 is fluorescence intensity in the resting cell. For the dual-fluorescence mode (Fig. 3), SBFI fluorescence was alternately excited at 340 (12 nm bandpass) and 380 (12) nm and collected at 510 (84) nm at the sampling frequency of 12.5 Hz (80

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ms per 340/380 nm image pair), using the same fluorescent setup as described for [Ca2⫹]i measurements. In this case, [Na⫹]i changes were presented as F340/F380 ratios. Fluorescence was corrected for background, as determined prior to the loading of cells with the indicator. Data are presented as mean⫾SEM. In order to elicit local Na⫹ entry within a small axonal region of a TTX-resistant DRG neuron, focal stimulation using an extracellular glass microelectrode was employed as described above for [Ca2⫹]i measurements. During focal stimulation action potentials were blocked by lowering extracellular Na⫹ concentration to 35 mM and adding 1 ␮M TTX (Medvedeva et al., 2008).

Reagents Bis-fura and SBFI were obtained from Invitrogen (Carlsbad, CA, USA); TTX and bicuculline were from Tocris (Ellisville, MO). All other reagents were purchased from Sigma (St. Louis, MO).

RESULTS Co-localization of Nav1.8 and Nav1.9 with presynaptic proteins in axonal boutons of DRG neurons The DRG/SC co-culture system has been established as an important tool to study the first sensory synapse under highly defined recording conditions (Gu and Macdermott, 1997; Vikman et al., 2001b; Sikand and Premkumar, 2007; Medvedeva et al., 2008). We used this co-culture system to examine the effects of TTX on Na⫹ and Ca2⫹ influx in single presynaptic boutons of DRG neurons and on sensory synaptic transmission. Using Nav1.8- and Nav1.9specific antibodies (Amaya et al., 2000; Fjell et al., 2000; Djouhri et al., 2003), we found extensive labeling of sensory axons and axonal boutons with a diameter of 1–2 ␮m and a length of 1– 4 ␮m (Fig. 1). Presynaptic boutons of similar size have been described on the central processes of primary afferent neurons in vivo (Maxwell and Rethelyi, 1987). The majority of the labeled axonal boutons also showed immunoreactivity for the presynaptic proteins synaptophysin (Fig. 1A, B; 152 of 166 Nav1.8-positive boutons, and 114 of 120 Nav1.9-positive boutons; four independent culture preparations obtained from 16 animals) and bassoon (Fig. 1C, D; 103 of 114 Nav1.8-positive boutons, and 71 of 75 Nav1.9-positive boutons; four independent culture preparations obtained from 16 animals). Examination of all synaptophysin and bassoon puncta showed that 152 of 287 synaptophysin puncta were immunoreactive for Nav1.8 and 114 of 236 synaptophysin puncta were immunoreactive for Nav1.9; similarly, 103 of 247 bassoon puncta were immunoreactive for Nav1.8 and 71 of 182 bassoon puncta were immunoreactive for Nav1.9. We also found that the majority of Nav1.8- and Nav1.9-positive boutons were associated with the postsynaptic density protein PSD95 (Fig. 1E, F; 109 of 128 Nav1.8positive boutons, and 74 of 88 Nav1.9-positive boutons; three independent culture preparations obtained from 12 animals). Overall, these data indicate that Nav1.8 and Nav1.9 are presynaptically expressed in a large proportion of DRG neurons. The presynaptic boutons that do not contain TTX-R Na⫹ channels likely belong to SC neurons and non-nociceptive DRG neurons. Expression of Nav1.8 and Nav1.9 starts at embryonic days 15 and 17, respectively,

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Fig. 1. Nav1.8 and Nav1.9 co-localize with the presynaptic proteins synaptophysin and bassoon in axonal boutons of DRG neurons. Distribution of Nav1.8 (red; A, C, E), Nav1.9 (red; B, D, F), synaptophysin (green; syn.; A, B), bassoon (green; C, D) and PSD95 (green; E, F) in DRG/SC co-cultures. The merged images demonstrate co-localization of the TTX-R Na⫹ channels with the synaptic proteins (yellow).

and increases during development reaching the adult levels by P7 (Benn et al., 2001). Given that we used cultures obtained from P0 –P2 rats, it is possible that in adult animals TTX-R Na⫹ channels are expressed even in a greater proportion of synapses than that found in our study. Presynaptic voltage-gated Naⴙ influx is resistant to TTX in a subset of DRG neurons Next, we examined the sensitivity of presynaptic Na⫹ influx to TTX by monitoring changes in the presynaptic Na⫹ concentration ([Na⫹]i) (Fig. 2). We used a method similar to the one we have previously developed for measuring presynaptic [Ca2⫹]i transients in DRG neurons (Usachev et al., 2002; Medvedeva et al., 2008). The fluorescent Na⫹ indicator SBFI was loaded via the patch pipette using the whole-cell patch clamp configuration, which enabled us to

visualize axons and axonal boutons (images in Figs. 2 and 3). Only cells that were able to generate all-or-none action potentials in the presence of 200 or 500 nM TTX (TTXresistant action potentials) were used for further analysis (Fig. 2, insets). Stimulation of DRG neurons using trains of action potentials (20 Hz for 25 s) induced a rapid increase in presynaptic [Na⫹]i (Fig. 2). Similar changes in presynaptic [Na⫹]i were evoked in the presence of 200 nM TTX (Fig. 2). The total relative change in SBFI fluorescence induced by stimulation was 13.9⫾1.3% under control conditions, and 12.9⫾1.1% in the presence of TTX (n⫽23 boutons/5 cells; P⫽0.22, paired Student’s t-test); the rate of [Na⫹]i rise in presynaptic boutons during the first 2 s of stimulation was 2.8⫾0.3 and 3.0⫾0.4%/s in the absence or presence of 200 nM TTX, respectively (n⫽23 boutons/5 cells; P⫽0.13, paired Student’s t-test).

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Fig. 2. Detection of TTX-resistant presynaptic Na⫹ influx in DRG neurons. Trains of action potentials (APs; arrows; 20 Hz, 25 s) evoke rapid [Na⫹]i elevation in SBFI-loaded presynaptic boutons (white boxes; image) both in control HH buffer (left) and in the presence of 200 nM TTX (right; TTX treatment was initiated 5 min prior to the recording). Action potentials were induced by 4 ms current injection pulses of 200 pA. [Na⫹]i changes in individual boutons are indicated by different numbers and colors. Insets show action potential recordings in the same neuron made in the absence (left) or presence of 200 nM TTX (right). For these recordings, action potentials were evoked by applying 4 ms current injection pulses of incremental value (50 pA increment; shown under the membrane potential traces).

We further characterized VGSC-mediated Na⫹ entry in TTX-resistant DRG neurons in response to trains of action potentials of various durations. To improve the resolution of the [Na⫹]i measurements, we employed the dual-wavelength (F340/F380) SBFI recording method for these experiments (see Experimental Procedures for details). The magnitude of Na⫹ entry was quantified as ⌬RSBFI⫽Rpeak– Rrest, where Rpeak and Rrest are the SBFI F340/F380 ratios under resting conditions and at the peak of the response, respectively. At least five action potentials applied at 10 Hz were required to induce detectable [Na⫹]i elevations in the presynaptic boutons of DRG neurons. Under control conditions, the amplitudes of presynaptic [Na⫹]i elevation (⌬RSBFI) induced by 5, 20 and 50 action potentials (10 Hz) were 0.042⫾0.006, 0.120⫾0.025 and 0.212⫾0.043, respectively (Fig. 3A; n⫽22 boutons/6 cells). Treatment with 500 nM TTX did not change the magnitude of the [Na⫹]i response in TTX-resistant DRG neurons (Fig. 3A). Specifically, the amplitudes of presynaptic [Na⫹]i elevations induced by 5, 20 and 50 action potentials in the presence of 500 nM TTX were 0.047⫾0.006 (n⫽22 boutons/6 cells; P⫽0.46, relative to control; paired Student’s t-test), 0.132⫾0.015 (n⫽22 boutons/6 cells; P⫽0.70, relative to control; paired Student’s t-test) and 0.280⫾0.016 (n⫽22 boutons/6 cells; P⫽0.21, relative to control; paired Student’s t-test), respectively. The described TTX-resistant [Na⫹]i elevations were abolished by treating cells with 5 mM lidocaine (n⫽22 boutons/6 cells) that blocks both TTX-S and TTX-R Na⫹ channels (Fig. 3A). Mapping of action potential-induced [Na⫹]i changes along the axon (Fig. 3B, C), showed that there was no delay in the [Na⫹]i elevation in boutons relative to the [Na⫹]i elevation in adjacent axonal segments. There was also no detectable difference in the amplitudes of the presynaptic and extrasynaptic [Na⫹]i elevations. Specifically, in the presence of 500 nM TTX the amplitudes of

extrasynaptic [Na⫹]i transients induced by 5, 20 and 50 action potentials were 0.048⫾0.004 (n⫽44 axonal segments/6 cells; P⫽0.91, relative to boutons; Student’s t-test), 0.128⫾0.010 (n⫽44 axonal segments/6 cells; P⫽0.82, relative to boutons; Student’s t-test) and 0.244⫾ 0.034 (n⫽44 axonal segments/6 cells; P⫽0.46, relative to boutons; Student’s t-test), respectively. These findings argue against the possibility that the TTX-resistant [Na⫹]i elevation in presynaptic boutons was caused by Na⫹ diffusion from the adjacent axonal areas. In contrast, imaging of [Na⫹]i in the cell somata revealed a prominent radial gradient of [Na⫹]i elevation directed away from the plasma membrane (Fig. 3D). In addition, we observed a strong and sustained [Na⫹]i gradient along the axon in response to a focal stimulation (Fig. 3E, F), suggesting that our method was sufficiently sensitive to detect non-uniform Na⫹ entry. In the latter case, localized Na⫹ entry through TTX-R Na⫹ channels was evoked by focal stimulation via a fine extracellular glass microelectrode positioned near the axon (see Experimental Procedures for details) while action potentials were blocked by lowering extracellular Na⫹ concentration to 35 mM and adding 1 ␮M TTX (Medvedeva et al., 2008). Under these conditions, the rise in [Na⫹]i was initially restricted to a small (⬍5 ␮m) axonal segment under the stimulating electrode, and then propagated in both directions along the axon. As shown in Fig. 3E and 3F, [Na⫹]i increase started with an 80 –160 ms delay in the areas located 3–5 ␮m away from the stimulating electrode, whereas for the axonal bouton located 10 ␮m from the electrode (area 1 in Fig. 3E) the delay was 160 –240 ms. Focal stimulation also led to a marked distance-dependent reduction in the amplitude of [Na⫹]i elevation along the axon. For example, for the areas located 3–5 ␮m and 8 –10 ␮m away from the stimulating electrode, the amplitude of [Na⫹]i response was only 71⫾5% and 50⫾2%, respec-

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Fig. 3. Characterization of TTX-resistant [Na⫹]i transients in axons and axonal boutons of DRG neurons. (A) [Na⫹]i elevations were recorded in two SBFI-loaded presynaptic boutons (white boxes; image) in response to 20 and 50 action potentials (APs; arrows) applied at 10 Hz under control conditions and in the presence of either 500 nM TTX or 5 mM lidocaine. Periods of drug application are indicated by horizontal bars under the traces. Pairs of 340/380 images were taken every 80 ms. Changes in [Na⫹]i are presented as F340/F380 ratios. Image on the left shows the distribution of SBFI fluorescence (␭ex⫽380 nm) in axons and axonal boutons of a patch-clamped DRG neuron at rest. (B) Action potential-induced [Na⫹]i elevations develop uniformly in axonal boutons (area 3/black) and adjacent axonal segments (areas 1/red, 2/green, 4/gray and 5/blue). These recordings were made from the cell shown in A. [Na⫹]i responses were elicited by the application of 20 (left) or 50 (right) action potentials (10 Hz) in the presence of 500 nM TTX using the whole-cell current-clamp configuration of patch clamp. Dotted lines show resting [Na⫹]i. Simultaneous recordings of action potentials are shown under the [Na⫹]i traces. (C) The distribution of [Na⫹]i in an axon and axonal bouton is presented using a color-coded F340/F380 scale for the experiment shown in B (right trace). The images depict the [Na⫹]i distribution at rest and at various times after initiation of stimulation (t⫽0 s) with 50 action potentials (10 Hz). (D) [Na⫹]i recording from the cell soma of the same DRG neuron as in A–C shows that [Na⫹]i elevation in the center of the cell was markedly smaller than that attained near the plasma membrane. The [Na⫹]i response was elicited by 200 action potentials (10 Hz; arrow) in the presence of 500 nM TTX. (E) Focal electrical stimulation induces localized Na⫹ entry and reveals [Na⫹]i gradient along the axon. Focal stimulation (5 ms pulses, four times, 10 Hz; arrows) was applied via an extracellular glass microelectrode (red dotted lines; image) positioned within 2–5 ␮m of a stimulated axonal segment. Action potentials were blocked by lowering extracellular Na⫹ concentration to 35 mM (equimolar replacement with choline-Cl) and by adding 1 ␮M TTX (Medvedeva et al., 2008). [Na⫹]i elevation was initially restricted to the stimulated region (area 3/red) and then, with some delay, spread to more distant axonal areas, including the axonal bouton (area 1, black). (F) The distribution of the axonal [Na⫹]i is presented using a color-coded F340/F380 scale, for a TTX-resistant DRG neuron at rest and at various times after initiation of focal stimulation (t⫽0 s). The images were obtained from the experiment shown in E. The position of the stimulating microelectrode is indicated by red dotted lines.

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tively, of those found in the axonal regions directly under the stimulating electrode (n⫽7 recordings/4 cells). Thus, our technique is sufficiently sensitive to resolve non-uniform Na⫹ entry along the axon, yet no delay or reduction in TTX-resistant Na⫹ entry has been detected in boutons relative to the adjacent axons in response to propagating action potentials. Taken together, the described data indicate that functional TTX-R Na⫹ channels are present in the presynaptic boutons of a subset of DRG neurons. Presynaptic [Ca2ⴙ]i elevation and sensory synaptic transmission can be retained in the presence of TTX Presynaptic VGSCs enable the propagation of action potentials into the presynaptic boutons, and this triggers voltage-gated Ca2⫹ influx and transmitter release (Engel and Jonas, 2005). Presynaptically expressed TTX-R Na⫹ channels are predicted to render presynaptic Ca2⫹ influx and transmitter release resistant to TTX. We tested this idea by performing Ca2⫹ imaging in TTX-resistant DRG neurons and by studying evoked synaptic responses. [Ca2⫹]i changes in presynaptic boutons were monitored as previously described (Usachev et al., 2002; Medvedeva et al., 2008). TTX resistance was defined by the ability of a neuron to generate action potentials in the presence of 200 or 500 nM TTX. As shown in Fig. 4A and 4B, delivery of a single action potential elicited a rapid elevation of presynaptic [Ca2⫹]i. Treatments with either 200 or 500 nM TTX did not block the responses, and presynaptic [Ca2⫹]i transients of similar size and duration were evoked by an action potential despite the presence of TTX (Fig. 4A, B). Under control conditions, the amplitude of presynaptic [Ca2⫹]i elevation (⌬RBis-Fura⫽Rpeak–Rrest, where Rpeak and Rrest are the bis-fura F340/F380 ratios under resting conditions and at the peak of the response, respectively) was 0.14⫾0.01 (n⫽37 boutons/9 cells); in the presence of 200 or 500 nM TTX the corresponding amplitudes were 0.16⫾0.01 (n⫽23 boutons/4 cells) and 0.14⫾0.02 (n⫽14 boutons/5 cells), respectively, which is not different from those obtained under control conditions (P⬎0.05, one-way ANOVA with Bonferroni’s post hoc test). We have also compared action potential–induced Ca2⫹ fluxes between boutons and the adjacent axonal areas in the presence of 500 nM TTX (Fig. 4C). We did not detect any delay in [Ca2⫹]i rise or reduction in the amplitude of [Ca2⫹]i in boutons relative to those observed in the adjacent axon (axonal ⌬RBis-Fura⫽0.12⫾0.02; 28 axonal segments/5 cells; P⫽0.46, relative to presynaptic boutons, Student’s t-test). This argues against the possibility that the presynaptic [Ca2⫹]i rise was caused by Ca2⫹ diffusion from the neighboring axonal regions. As in the case of the [Na⫹]i measurements, we examined whether our method is sufficiently sensitive to detect [Ca2⫹]i gradients along the axon that are caused by nonuniform Ca2⫹ entry. In this case, [Ca2⫹]i elevation was induced by focal stimulation of a small axonal area under conditions when action potentials were blocked (Fig. 4D; see Experimental Procedures for details). Such stimulation led to a [Ca2⫹]i rise that was restricted to a small axonal

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area directly under the stimulating electrode. We observed a significant distance-dependent decrement in the amplitude of the [Ca2⫹]i increase (Fig. 4D). For example, [Ca2⫹]i elevations monitored in regions 3–5 and 8 –10 ␮m distant from the electrode constituted only 56⫾9% and 27%⫾8%, respectively, of those found in the axonal segments near the stimulating electrode (n⫽6 recordings/4 cells). Thus, our method is sufficiently sensitive to detect [Ca2⫹]i gradients along the axon that are generated by non-uniform Ca2⫹ entry. Collectively, our findings suggest that the observed action potential–induced TTX-resistant presynaptic [Ca2⫹]i elevation is a direct result of Ca2⫹ entry into the presynaptic bouton, rather than a consequence of diffusion from the adjacent axonal regions. Our findings that action potential–induced Na⫹ and 2⫹ Ca influxes are resistant to TTX in a large proportion of axons and axonal boutons predict that glutamatergic synaptic transmission would also be resistant to TTX in a subset of sensory synapses formed between DRG and SC neurons. Indeed, we found that glutamatergic synaptic responses between DRG and SC neurons could be evoked in the presence of 200 nM TTX (Fig. 5) in 8 of 14 synaptic pairs tested. For these TTX-resistant synapses, the EPSC amplitudes were 302⫾58 and 254⫾56 pA in the absence (control) or presence of 200 nM TTX, respectively (n⫽8; P⫽0.26, paired Student’s t-test). The EPSC latencies were 5.4⫾1.2 ms in control and 8.1⫾1.5 ms in the presence of TTX (n⫽8; P⫽0.15, paired Student’s t-test). In some experiments, we tested the TTX resistance of synaptic responses using higher concentrations of the drug, 500 nM TTX. In this latter case, 5 synaptic pairs of 12 tested were resistant to 500 nM TTX. The EPSC amplitudes were 270⫾82 pA under control conditions and 224⫾64 pA in the presence of 500 nM TTX (n⫽5; P⫽0.70, paired Student’s t-test); the EPSC latencies were 5.4⫾0.6 and 9.9⫾2.0 ms under control conditions and in the presence of 500 nM TTX, respectively (n⫽5; P⫽0.11, paired Student’s t-test). These TTX-resistant EPSCs were completely blocked by the addition of 5 mM lidocaine in all synaptic pairs tested (Fig. 5; n⫽11).

DISCUSSION Overall, our results provide strong evidence for presynaptic expression of functional TTX-R Na⫹ channels. First, we showed co-localization of both Nav1.8 and Nav1.9 with two different presynaptic proteins, synaptophysin and bassoon, in axonal boutons of a subset of DRG neurons. Most of the Nav1.8- and Nav1.9-positive boutons were found in association with the postsynaptic protein PSD95. Second, we directly demonstrated action potential– evoked presynaptic Na⫹ influx that was resistant to TTX. The fact that the rate and the size of presynaptic [Na⫹]i rise were not significantly affected by the TTX treatment suggests that TTX-R Na⫹ channels are the predominant VGSCs in presynaptic boutons of TTX-resistant DRG neurons. Finally, we found that presynaptic Ca2⫹ influx and EPSCs were not blocked by TTX in a subset of sensory synapses.

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Fig. 4. Presynaptic [Ca2⫹]i elevation is not blocked by TTX in DRG neurons expressing TTX-R VGSCs. (A) To facilitate visualization of presynaptic boutons DRG neurons were transfected with synaptophysin-EGFP (Syn-EGFP) using lentivirus, and subsequently were loaded with bis-fura as described in the Experimental Procedures. Action potentials (APs) were resistant to 200 nM TTX in this cell (not shown). Images depict the axonal distribution of bis-fura (␭ex⫽380 nm) and Syn-EGFP (␭ex⫽475 nm). Traces show [Ca2⫹]i elevations in individual presynaptic boutons (white boxes in bis-fura image) in response to a single action potential (arrow) in the absence (left) or presence of 200 nM TTX (right; 5 min pretreatment). Action potentials were evoked using a glass extracellular stimulating electrode positioned near the cell body of a DRG neuron, as described in Experimental Procedures. (B) TTX-resistant presynaptic [Ca2⫹]i changes in response to a single action potential were recorded using the current-clamp mode of patch clamp. Image (left) shows axonal distribution of bis-fura fluorescence (␭ex⫽380 nm) and the areas (white boxes) from which [Ca2⫹]i recordings were made. Traces (right) show [Ca2⫹]i elevations in axonal boutons (areas 3/black and 6/red) in response to a single action potential evoked in controls and in the presence of 500 nM TTX. The corresponding action potentials are shown in the insets. (C) Comparison of [Ca2⫹]i elevation in the axonal bouton (area 3/black; see image in B) with those observed in the adjacent axonal regions (areas 1/green, 2/gray, 4/red and 5/blue) for the experiment shown in B. Ca2⫹ entry was evoked by a single action potential (arrow) in the presence of 500 nM TTX. (D) Focal stimulation induces a steep [Ca2⫹]i gradient along the axon. Focal stimulation was performed using an extracellular glass microelectrode (dotted red lines) positioned within

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Fig. 5. Sensory synaptic transmission can be retained in the presence of TTX. The graph shows a representative time course of evoked EPSC amplitudes for a TTX-resistant synaptic pair. The timing of application of TTX (200 nM) and lidocaine (5 mM) is indicated by horizontal bars. Representative EPSC traces obtained under each condition are shown above the plot.

We studied the first sensory synapse using the DRG/ SC co-culture system. This system enabled us to examine presynaptic Na⫹ and Ca2⫹ influx simultaneously with membrane potential recordings in single identified DRG neurons under the conditions when the cell exposure to TTX and other drugs was precisely controlled. The work by others and by our group showed that most characteristics of sensory synapses in the DRG/SC co-culture are similar to those found in vivo, including electrophysiological and pharmacological properties, as well as the spectrum of the pre- and postsynaptic ion channels and receptors (Gruner and Silva, 1994; Gu and Macdermott, 1997; Vikman et al., 2001a; Lee et al., 2004; Tsuzuki et al., 2004; Sikand and Premkumar, 2007; Medvedeva et al., 2008). Furthermore, complex phenomena such as windup and long-term potentiation have also been described in a DRG/SC co-culture (Vikman et al., 2001b). Nonetheless, one has to acknowledge the limitations of this co-culture system which include the lack of complex laminar organization of the neuronal network characteristic of the dorsal horn, the difference in the repertoire of glial and other nonneuronal cells between the in vivo and in vitro conditions, and the influence of the culture medium composition. Although our conclusions will have to be ultimately tested in vivo, we note that the available data obtained in more intact systems are in good agreement with our findings. Specifically, Jeftinija (1994) demonstrated TTX-resistant sensory synaptic transmission in SC slices. In addition, two studies reported Nav1.8 and Nav1.9 immunoreactivities in

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the dorsal horn of the SC suggesting expression of the channels in the central terminals of primary afferent neurons (Novakovic et al., 1998; Amaya et al., 2000). We now show for the first time co-localization of Nav1.8 and Nav1.9 with synaptic markers and directly demonstrate presynaptic TTX-resistant Na⫹ influx in DRG neurons. Axonal and synaptic distribution of VGSCs varies significantly among the synapses. Examination of presynaptic currents at the mouse neuromuscular junction has shown that VGSCs are present in the preterminal part but are excluded from the presynaptic area (Brigant and Mallart, 1982). VGSCs are also absent from the presynaptic terminals of the calyx of Held synapse as has been recently demonstrated by using patch-clamp recordings and confocal microscopy (Leao et al., 2005). In these synapses, the spread of depolarization from parent axon into the active zone is likely a passive process. In contrast, a high density of VGSCs was detected in hippocampal mossy fiber boutons (Engel and Jonas, 2005). It has been proposed that axonal and presynaptic VGSCs have different roles in the regulation of synaptic transmission. While axonal VGSCs control the reliability and speed of action potential propagation, presynaptic VGSCs can influence presynaptic Ca2⫹ signaling and transmitter release via at least two mechanisms. First, they can amplify presynaptic action potential and voltage-gated Ca2⫹ entry (Engel and Jonas, 2005). Second, their activity can lead to accumulation of presynaptic Na⫹, and this might slow the clearance of presynaptic Ca2⫹ by the plasma membrane Na⫹/ Ca2⫹ exchanger (Leao et al., 2005). It is likely that presynaptic TTX-R Na⫹ channels influence transmitter release at sensory synapses through similar mechanisms, especially given their critical role in shaping action potentials in nociceptors (Renganathan et al., 2001; Blair and Bean, 2002). A relative resistance of Nav1.8 to inactivation at depolarized membrane potential is particularly important, as this property of Nav1.8 enables DRG neurons expressing these channels to maintain large amplitude of action potentials under the conditions of sustained depolarization caused by extensive neuronal activity and accumulation of extracellular K⫹ (Harty and Waxman, 2007). Moreover, the contribution of presynaptic TTX-R channels can potentially be enhanced by prostaglandin E2 and other pain mediators that modulate Nav1.8 and Nav1.9 and are produced in the SC following peripheral injury or inflammation (Gold et al., 1996; Svensson and Yaksh, 2002; Rush and Waxman, 2004). This idea would be consistent with the finding that intrathecal administration of a selective Nav1.8 inhibitor, ␮O-conotoxin MrVIB, reduces inflammatory pain (Ekberg et al., 2006). Pharmacological studies also suggest that Nav1.8 expressed in the SC contributes to neuropathic pain (Ekberg

2–5 ␮m of the axonal segment under examination. Action potentials were blocked by lowering extracellular Na⫹ concentration to 35 mM (equimolar replacement with choline-Cl) and by adding 1 ␮M TTX (Medvedeva et al., 2008). Traces (right) show [Ca2⫹]i changes in the stimulated axonal segment (area 3/red) and in other adjacent axonal regions (areas 1/green, 2/gray, 4/black and 5/blue) in response to one or two electrical stimuli (2 ms each; arrows). The [Ca2⫹]i response was largest in the area under the stimulating electrode, and decreased in size dramatically as the distance from the stimulating electrode increased. For example, the amplitude of the [Ca2⫹]i elevation in an axonal bouton (area 4/black) located ⬃4 ␮m from the stimulating electrode was ⬍50% of that in the area under the electrode (area 3/red).

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et al., 2006; McGaraughty et al., 2008). The function of Nav1.9 in the SC remains to be examined. Future studies employing selective genetic and pharmacological tools are needed to define the specific role of presynaptic Nav1.8 and Nav1.9 channels in spinal pain processing following inflammation and nerve injury. Acknowledgments—This work was supported by National Institutes of Health grant NS054614 and American Heart Association National Scientist Development Grant to Y.M.U. We thank Donna Hammond for helpful comments on this manuscript, John Wood for the gift of Nav1.8 antibodies and Jane Sullivan for the gift of the synaptophysin-EGFP plasmid.

REFERENCES Akopian AN, Sivilotti L, Wood JN (1996) A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 379:257–262. Akopian AN, Souslova V, England S, Okuse K, Ogata N, Ure J, Smith A, Kerr BJ, McMahon SB, Boyce S, Hill R, Stanfa LC, Dickenson AH, Wood JN (1999) The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat Neurosci 2:541–548. Amaya F, Decosterd I, Samad TA, Plumpton C, Tate S, Mannion RJ, Costigan M, Woolf CJ (2000) Diversity of expression of the sensory neuron-specific TTX-resistant voltage-gated sodium ion channels SNS and SNS2. Mol Cell Neurosci 15:331–342. Amaya F, Wang H, Costigan M, Allchorne AJ, Hatcher JP, Egerton J, Stean T, Morisset V, Grose D, Gunthorpe MJ, Chessell IP, Tate S, Green PJ, Woolf CJ (2006) The voltage-gated sodium channel Na(v)1.9 is an effector of peripheral inflammatory pain hypersensitivity. J Neurosci 26:12852–12860. Benn SC, Costigan M, Tate S, Fitzgerald M, Woolf CJ (2001) Developmental expression of the TTX-resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS2) in primary sensory neurons. J Neurosci 21:6077– 6085. Bhave G, Gereau RW 4th (2004) Posttranslational mechanisms of peripheral sensitization. J Neurobiol 61:88 –106. Blair NT, Bean BP (2002) Roles of tetrodotoxin (TTX)-sensitive Na⫹ current, TTX-resistant Na⫹ current, and Ca2⫹ current in the action potentials of nociceptive sensory neurons. J Neurosci 22: 10277–10290. Brigant JL, Mallart A (1982) Presynaptic currents in mouse motor endings. J Physiol 333:619 – 636. Cummins TR, Sheets PL, Waxman SG (2007) The roles of sodium channels in nociception: implications for mechanisms of pain. Pain 131:243–257. Cummins TR, Waxman SG (1997) Downregulation of tetrodotoxinresistant sodium currents and upregulation of a rapidly repriming tetrodotoxin-sensitive sodium current in small spinal sensory neurons after nerve injury. J Neurosci 17:3503–3514. Dib-Hajj SD, Tyrrell L, Black JA, Waxman SG (1998) NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and downregulated after axotomy. Proc Natl Acad Sci U S A 95:8963– 8968. Djouhri L, Fang X, Okuse K, Wood JN, Berry CM, Lawson SN (2003) The TTX-resistant sodium channel Nav1.8 (SNS/PN3): expression and correlation with membrane properties in rat nociceptive primary afferent neurons. J Physiol Lond 550:739 –752. Ekberg J, Jayamanne A, Vaughan CW, Aslan S, Thomas L, Mould J, Drinkwater R, Baker MD, Abrahamsen B, Wood JN, Adams DJ, Christie MJ, Lewis RJ (2006) muO-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits. Proc Natl Acad Sci U S A 103:17030 –17035.

Engel D, Jonas P (2005) Presynaptic action potential amplification by voltage-gated Na⫹ channels in hippocampal mossy fiber boutons. Neuron 45:405– 417. Fang X, Djouhri L, Black JA, Dib-Hajj SD, Waxman SG, Lawson SN (2002) The presence and role of the tetrodotoxin-resistant sodium channel Nav1.9 (NaN) in nociceptive primary afferent neurons. J Neurosci 22:7425–7433. Fjell J, Hjelmstrom P, Hormuzdiar W, Milenkovic M, Aglieco F, Tyrrell L, Dib-Hajj S, Waxman SG, Black JA (2000) Localization of the tetrodotoxin-resistant sodium channel NaN in nociceptors. Neuroreport 11:199 –202. Gold MS, Reichling DB, Shuster MJ, Levine JD (1996) Hyperalgesic agents increase a tetrodotoxin-resistant Na⫹ current in nociceptors. Proc Natl Acad Sci U S A 93:1108 –1112. Gold MS, Weinreich D, Kim C-S, Wang R, Treanor J, Porreca F, Lai J (2003) Redistribution of NaV1.8 in uninjured axons enables neuropathic pain. J Neurosci 23:158 –166. Gruner W, Silva LR (1994) Omega-conotoxin sensitivity and presynaptic inhibition of glutamatergic sensory neurotransmission in vitro. J Neurosci 14:2800 –2808. Gu JGG, Macdermott AB (1997) Activation of ATP P2x receptors elicits glutamate release from sensory neuron synapses. Nature 389:749 –753. Harty PT, Waxman SG (2007) Inactivation properties of sodium channel Nav1.8 maintain action potential amplitude in small DRG neurons in the context of depolarization. Mol Pain 3:1–9. Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF, Kort M, Carroll W, et al. (2007) A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc Natl Acad Sci U S A 104:8520 – 8525. Jeftinija S (1994) The role of tetrodotoxin-resistant sodium channels of small primary afferent fibers. Brain Res 639:125–134. Jin X, Gereau RW 4th (2006) Acute p38-mediated modulation of tetrodotoxin-resistant sodium channels in mouse sensory neurons by tumor necrosis factor-alpha. J Neurosci 26:246 –255. Johnston JC, Gasmi M, Lim LE, Elder JH, Yee JK, Jolly DJ, Campbell KP, Davidson BL, Sauter SL (1999) Minimum requirements for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors. J Virol 73:4991–5000. Kirischuk S, Veselovsky N, Grantyn R (1999) Relationship between presynaptic calcium transients and postsynaptic currents at single gamma-aminobutyric acid (GABA)ergic boutons. Proc Natl Acad Sci U S A 96:7520 –7525. Kostyuk PG, Veselovsky NS, Tsyndrenko AY (1981) Ionic currents in the somatic membrane of rat dorsal root ganglion neurons-I. Sodium currents. Neuroscience 6:2423–2430. Lai J, Gold MS, Kim CS, Bian D, Ossipov MH, Hunter JC, Porreca F (2002) Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8. Pain 95: 143–152. Lai J, Porreca F, Hunter JC, Gold MS (2004) Voltage-gated sodium channels and hyperalgesia. Annu Rev Pharmacol Toxicol 44:371–397. Leao RM, Kushmerick C, Pinaud R, Renden R, Li GL, Taschenberger H, Spirou G, Levinson SR, Gersdorff VH (2005) Presynaptic Na⫹ channels: locus, development, and recovery from inactivation at a high-fidelity synapse. J Neurosci 25:3724 –3738. Lee CJ, Labrakakis C, Joseph DJ, Macdermott AB (2004) Functional similarities and differences of AMPA and kainate receptors expressed by cultured rat sensory neurons. Neuroscience 129:35–48. Maxwell D, Rethelyi M (1987) Ultrastructure and synaptic connections of cutaneous afferent fibers in the spinal cord. Trends Neurosci 10:117–123. McGaraughty S, Chu KL, Scanio MJ, Kort ME, Faltynek CR, Jarvis MF (2008) A selective Nav1.8 sodium channel blocker, A-803467 [5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide], attenuates spinal neuronal activity in neuropathic rats. J Pharmacol Exp Ther 324:1204 –1211.

Y. V. Medvedeva et al. / Neuroscience 159 (2009) 559 –569 Medvedeva YV, Kim MS, Usachev YM (2008) Mechanisms of prolonged presynaptic Ca2⫹ signaling and glutamate release induced by TRPV1 activation in rat sensory neurons. J Neurosci 28: 5295–5311. Novakovic SD, Tzoumaka E, McGivern JG, Haraguchi M, Sangameswaran L, Gogas KR, Eglen RM, Hunter JC (1998) Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions. J Neurosci 18:2174 –2187. Pinto V, Derkach VA, Safronov BV (2008) Role of TTX-sensitive and TTX-resistant sodium channels in Adelta- and C-fiber conduction and synaptic transmission. J Neurophysiol 99:617– 628. Priest BT, Murphy BA, Lindia JA, Diaz C, Abbadie C, Ritter AM, Liberator P, Iyer LM, Kash SF, Kohler, MG, Kaczorowski GJ, MacIntyre DE, Martin WJ (2005) Contribution of the tetrodotoxinresistant voltage-gated sodium channel NaV1.9 to sensory transmission and nociceptive behavior. Proc Natl Acad Sci U S A 102:9382–9387. Quasthoff S, Grosskreutz J, Schroder JM, Schneider U, Grafe P (1995) Calcium potentials and tetrodotoxin-resistant sodium potentials in unmyelinated C fibres of biopsied human sural nerve. Neuroscience 69:955–965. Renganathan M, Cummins TR, Waxman SG (2001) Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons. J Neurophysiol 86:629 – 640. Roza C, Laird JM, Souslova V, Wood JN, Cervero F (2003) The tetrodotoxin-resistant Na⫹ channel Nav1.8 is essential for the expression of spontaneous activity in damaged sensory axons of mice. J Physiol Lond 550:921–926. Rush AM, Waxman SG (2004) PGE2 increases the tetrodotoxin-resistant Nav1.9 sodium current in mouse DRG neurons via G proteins. Brain Res 1023:264 –271.

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Sikand P, Premkumar LS (2007) Potentiation of glutamatergic synaptic transmission by protein kinase C-mediated sensitization of TRPV1 at the first sensory synapse. J Physiol Lond 581:631– 647. Svensson CI, Yaksh TL (2002) The spinal phospholipase-cyclooxygenase-prostanoid cascade in nociceptive processing. Annu Rev Pharmacol Toxicol 42:553–583. Tate S, Benn S, Hick C, Trezise D, John V, Mannion RJ, Costigan M, Plumpton C, Grose D, Gladwell Z, Kendall G, Dale K, Bountra C, Woolf CJ (1998) Two sodium channels contribute to the TTX-R sodium current in primary sensory neurons. Nat Neurosci 1: 653– 655. Tsuzuki K, Xing H, Ling J, Gu JG (2004) Menthol-induced Ca2⫹ release from presynaptic Ca2⫹ stores potentiates sensory synaptic transmission. J Neurosci 24:762–771. Usachev YM, DeMarco SJ, Campbell C, Strehler EE, Thayer SA (2002) Bradykinin and ATP accelerate Ca2⫹ efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca2⫹ pump isoform 4. Neuron 33:113–122. Vikman KS, Backstrom E, Kristensson K, Hill RH (2001a) A twocompartment in vitro model for studies of modulation of nociceptive transmission. J Neurosci Methods 105:175–184. Vikman KS, Kristensson K, Hill RH (2001b) Sensitization of dorsal horn neurons in a two-compartment cell culture model: wind-up and long-term potentiation-like responses. J Neurosci 21:RC169. Wood JN, Boorman JP, Okuse K, Baker MD (2004) Voltage-gated sodium channels and pain pathways. J Neurobiol 61:55–71. Yoshimura M, Jessell T (1990) Amino acid-mediated EPSPs at primary afferent synapses with substantia gelatinosa neurones in the rat spinal cord. J Physiol Lond 430:315–335.

(Accepted 16 December 2008) (Available online 30 December 2008)