Action potentials in masseter afferent neurons were examined in whole cell current clamp recordings (Figure 1). Neurons were held at -70 mV and sufficient current injected (0.5–3 ms duration) to elicit an action potential. Action potential duration was measured at 0 mV. Action potentials elicited from masseter afferents isolated from the TG varied considerably in width, with durations between 0.6 ms and 5.2 ms at 0 mV (average duration 2.2 ± 0.2 ms, n = 27, Figure 1B). By contrast, MeV masseter afferent action potentials were narrower and displayed much less variability with a mean AP duration of 0.8 ± 0.05 ms (range 0.6 ms to 1.1 ms, n = 13, Figure 1B).

The types of voltage-dependent sodium channel currents (I_Na) contributing to the action potentials in masseter afferents were examined using conventional voltage clamp recordings made in low extracellular Na (20 mM or 40 mM) in order to minimize the size of evoked I_Na. The peak I_Na was determined by stepping the neurons from a holding potential of -90 mV to potentials between -100 mV and +45 mV. Even in conditions of reduced external Na, the peak amplitude of evoked currents was substantial; peak I_Na averaged 11 ± 3 nA in ganglion afferents (n = 27) and 22 ± 4 nA in masseter afferents isolated from the MeV nucleus (n = 15). Application of tetrodotoxin (TTX, 300 nM), a potent inhibitor of many types of I_Na, inhibited the peak I_Na in ganglion masseter afferents in a highly variable manner (Figure 2). The average inhibition by TTX was 38 ± 6% (n = 27) but the inhibition ranged in individual cells from nothing to 100%. In contrast, TTX completely inhibited the peak I_Na in masseter afferents isolated from the MeV nucleus (99 ± 1%, n = 15, Figure 2).

The calcium channel current (I_Ca) density of neurons was determined by repetitively stepping the membrane potential from a holding potential of -90 mV to test potentials between -60 and +60 mV. The I_Ca density of masseter afferents was similar to that of unlabeled cells (115 ± 6 pA/pF, n = 104 vs 109 ± 9 pA/pF, n = 53). The calcium channel density of masseter afferents isolated from the MeV nucleus was 36 ± 8 pA/pF (n = 12).

The sensitivity of the masseter afferents to opioids was determined by examining the modulation of I_Ca by agonists selective for μ-, δ- and κ-opioid receptors and the nociceptin receptor ORL1 (Figure 3). Cells were considered responsive if there was a reversible inhibition of the I_Ca evoked by step from -90 mV to 0 mV of at least 10%. Maximally effective concentrations of the μ-opioid agonist DAMGO (3 μM–10 μM, 8) inhibited I_Ca in 74 of 92 labeled TG neurons (80%), and in 29 of 49 unlabeled neurons assessed at the same time (59%; P < 0.01, χ 2). In responsive masseter afferents DAMGO (3–10 μM) inhibited I_Ca by 46 ± 3%, in responsive unlabeled cells the inhibition of I_Ca was 39 ± 4%. We have previously arbitrarily divided trigeminal neurons into small (diameter <30 μm), intermediate (diameter between 30 μm–40 μm) and large (diameter >40 μm) cells 8. DAMGO inhibited I_Ca in 85% of small masseter afferents, in 73% of intermediate diameter cells and in 82% of large cells (Figure 3C). The inhibition of I_Ca by DAMGO was 48 ± 4% in small masseter afferents, 50 ± 4% in intermediate cells and 36 ± 4% in large masseter afferents. Neither the κ-opioid agonist U69-593 (3 μM, n = 10) or the δ-opioid agonist deltorphin II (n = 13) inhibited I_Ca in masseter afferents. U69-593 inhibited I_Ca in 1 of 11 unlabeled cells tested (by 57%), deltorphin II did not inhibit I_Ca in any unlabeled cells (n = 11). The ORL1 agonist nociceptin (300 nM-1 μM) inhibited I_Ca in 6/15 (40%) of masseter afferents (inhibition of I_Ca was 41 ± 9%) and in 5/12 (42%) of unlabeled cells (inhibition of I_Ca was 38 ± 12%). The GABA_B receptor agonist baclofen inhibited I_Ca in most ganglion masseter afferents (31/35; 89%, inhibition of I_Ca was 26 ± 4%) and unlabeled neurons (15/20; 75%, inhibition of I_Ca was 24 ± 4%).

We have previously reported that somatostatin receptors preferentially inhibit I_Ca in large tooth pulp nociceptors when compared with small nociceptors 8. Somatostatin inhibited I_Ca in 11/39 labelled masseter afferents and 3/15 unlabelled afferents. Somatostatin inhibited I_Ca in 5/19 small masseter afferents and 3/7 large masseter afferents (P = 0.4, χ2). The vast majority of masseter afferents that did (8/11) or did not (22/28) respond to SRIF also responded to DAMGO.

DAMGO (3 μM) inhibited I_Ca in only 1/11 MeV masseter afferents examined (by 18%). Baclofen (30 μM–100 μM) inhibited I_Ca in all MeV masseter afferents examined, by an average of 33 ± 2% (n = 10, Figure 3B).

We were able to examine the co-expression of all 3 of the putative nociception-related ion channels in 55 TG masseter afferents (Figure 5). Only 4/55 (7%) of neurons failed to express one of either TRPV1, ASIC or P2X₃-like current (Figure 5a). Conversely, only 3/55 (5%) of cells expressed all 3 types of current. About half the neurons (29/55, 53%) expressed at least 2 of the channels while the remainder (22/55, 40%) expressed only one of the nociception-related channels. The data for co-expression of channels is illustrated in Figure 5b. ASIC3-like currents were the most commonly co-expressed channel in neurons expressing TRPV1 or P2X₃-like currents, while no neurons expressed only TRPV1 and P2X₃-like currents.

The action potentials of the MeV masseter afferents were narrow and lacked an inflection on the downward component of the current, consistent with previous recordings from acutely isolated MeV neurons 22 and MeV neurons in brain slices 2324. The I_Na recorded from MeV masseter afferents were completely blocked by TTX. These data are consistent with reports that muscle spindle afferents do not express detectable Na_V1.8 or Na_V1.9 immunoreactivity 1011. The narrow action potentials of proprioceptive afferents are consistent with the very rapid firing rates that these neurons achieve-exceeding 200 Hz (e.g. 25). By contrast, the masseter afferents isolated from the TG had a wide range of action potential widths and shapes, and most cells had a significant component of TTX-resistant I_Na. TTX-resistant I_Na are subject to acute regulation by a variety sensory mediators acting via G protein-coupled or tyrosine kinase-linked receptors, particularly prostaglandins, bradykinin and nerve growth factor 26. The changes in I_Na availability produced by these mediators mean that afferents expressing TTX-resistant I_Na are likely to be subject to rapid changes in excitability reflecting the state of the tissue they innervate. Wider action potentials and greater amounts of TTX-resistant I_Na are strongly correlated with a nociceptive modality, but these properties vary between afferents of different conduction velocity classes as well as between afferents of different modality within a class 27, and one cannot define a neuron as nociceptive simply on the basis of a action potential duration or its sensitivity to TTX. Nevertheless, within the TG masseter afferents, which presumably contain cells with a nociceptive function, smaller neurons tended to have wider action potentials. There was no such relationship apparent within the proprioceptive MeV masseter afferents.

We found a wide variety of ATP-induced currents in TG masseter afferents, similar to results from other studies in sensory neurons 282930. Messenger RNA and receptor-like immunoreactivity for 6 of the 7 cloned subtypes of P2X receptor are found in the trigeminal ganglion 3132 and the currents we recorded are likely to be comprised of a mixture of homo- and heteromeric P2X receptor channels. Although attempting to define the P2X subunits responsible for the variety of ATP currents was beyond the scope of this study (but see 28) we attributed the rapidly desensitizing ATP current observed in some masseter afferents to P2X₃ receptor activation. Rapidly desensitizing ATP currents in sensory neurons have been reported to depend on the presence of the P2X₃ gene or have been identified pharmacologically as P2X₃ receptors 333435 and although the kinetically similar P2X₁ receptor has been shown to be present in sensory neurons by immunohistochemical methods, there is little electrophysiological evidence for currents mediated by P2X₁ receptors in rat or mouse sensory neurons 429.

P2X₃ subunits make a major contribution to the ATP currents in trigeminal neurons projecting to the tooth pulp, both as P2X₃ receptor homomers and putative P2X₂/P2X₃ heteromers 4. Relatively fewer masseter afferents express P2X₃-like immunoreactivity 36. In the present study we observed rapidly desensitizing ATP currents either alone or in combination with other P2X currents in about 55% of TG masseter afferents, which is similar to the proportion of tooth pulp nociceptors which displayed fast ATP currents (44%, 4). P2X₃-like immunoreactivity has been reported in about 25% of masseter afferents 36, which is a considerably smaller proportion than suggested by the present report. These differing results may reflect differing sensitivities of immunohistochemistry and electrophysiology, they may be due to the presence of some P2X₁-containing currents in masseter afferents or perhaps arise from the short time the isolated neurons spend in culture. 30 – 40% of tooth pulp afferents can be labeled with P2X₃ antiserum 437 but it is interesting to note that more than 50% of tooth pulp afferents challenged with ATP also displayed persistent currents. This indicates that while P2X₃-containing ATP receptors are found in many putative nociceptors, they are not the only P2X subunits that may detect noxious stimuli signalled by ATP.

The relatively high μ-opioid receptor sensitivity of jaw muscle afferents is similar to that reported in afferents projecting to hindlimb muscles, where about 75% of cells were sensitive to DAMGO 38. The apparently high sensitivity of muscle afferent I_Ca to μ-opioid agonists contrasts with the reported low frequency of μ-opioid agonist modulation of I_Ca in skin and colonic afferents (approximately 10%, 39). The relative insensitivity of MeV I_Ca to DAMGO (1 of 11 cells responding) is consistent with the extremely low mRNA abundance in these cells (2 of 72, 40). The modulation of I_Ca in MeV neurons by the GABA_B receptor agonist baclofen is in contrast to the lack of affect of baclofen on the membrane properties of MeV neurons in slices 41.

Interestingly, the μ-opioid receptor sensitivity of masseter muscle afferents differs markedly from that reported for the "purely nociceptive" afferents from tooth pulp 840. DAMGO inhibited I_Ca in more masseter afferents than tooth pulp afferents, regardless of cell size (80% versus 42% respectively 40), and most strikingly, DAMGO was equally effective in small and large masseter afferents (85% and 82% of cells inhibited respectively). By contrast, DAMGO inhibited I_Ca in only 30% of large tooth pulp afferents 8. As 30% of large masseter afferents had substantial capsaicin currents (> 500 pA), indicating that these cells are likely to be nociceptors, our data suggest that μ-opioid receptors may be differentially expressed in distinct populations of nociceptors projecting to different tissues in the head, i.e. preferentially expressed in muscle nociceptors versus tooth pulp nociceptors. These data suggest that opioid analgesics may be better at relieving some types of head pain than others, and further that the endogenous opioid analgesic systems of the periphery may display differential effectiveness against nociceptive stimuli arising from distinct structures. The apparently high expression of opioid receptors in TG masseter afferents also suggests that these receptors may have functions in muscle physiology other than simple inhibition of nociception.

All experiments were carried out using protocols approved by the OHSU Institutional Animal Care and Use Committee. Masseter afferents were labeled as outlined in detail in 42. Briefly, male Sprague-Dawley rats between 5–8 weeks old were anesthetized with a s.c. injection of "rat cocktail" consisting of ketamine (55 mg kg ^-1), xylazine (5.5 mg kg ^-1 and acepromazine (1.1 mg kg ^-1). A small incision was made in skin overlying each masseter muscle and 5 × 1 μl injections of DiI (5% in DMSO) were made into each muscle with a Hamilton 10 μl syringe. The wound was closed with cyanoacrylate glue and the animals returned to their cages. Sensory neurons were isolated 2 weeks after surgery.

Cells were isolated from trigeminal ganglia essentially as described in 42. Briefly, rats were anaesthetized with halothane (4%), and killed by decapitation. The trigeminal ganglia were removed and placed in cold Ca²⁺/Mg²⁺-Free Hanks Solution (CMF Hanks). Ganglia were cut up with iridectomy scissors and incubated at 35°C for 20 minutes in CMF Hanks plus papain (20 U ml^-1), followed by 20 minutes in CMF Hanks plus dispase (4 mg ml^-1) and collagenase (3 mg ml^-1). The enzyme incubation was stopped with F-12 media supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin/streptomycin, and the cells were released by gentle trituration through decreasing bore Pasteur pipettes with fire-polished tips. Cells were plated on plastic culture dishes precoated with poly-D-lysine and laminin. After the cells had settled they were cultured in a humidified chamber at room temperature in Leibovitz's L-15 medium supplemented with 10% FBS, 50 ng ml^-1 NGF, 5 mM glucose, 5 mM NaHEPES and 50 U ml^-1 penicillin/streptomycin.

Cells were isolated from the MeV nucleus by a modification of the methods outlined in 42. Briefly, rats were anaesthetized with halothane and killed by a blow to the chest. The brain was rapidly removed and a block containing the brainstem immersed in ice cold artificial cerebrospinal fluid of composition (mM): NaCl 126, KCl 2.5, NaH₂PO₄ 1.4, MgCl₂ 1.2, CaCl₂ 2.4, glucose 11, NaHCO₃ 25. Slices of the brainstem (400 μM) containing the MeV region were made with a Leica vibratome and the region containing the MeV cells subdissected with fine needles. The tissue chunks were placed in modified HBS (Solution 1 containing 10 mM MgCl₂, 2 mM CaCl₂) containing papain, 20 U ml^-1 and incubated for 3–5 minutes at 37°C. The enzyme incubation was stopped with F-12 media supplemented with 10% FBS, 25 ng ml ^-1 NT-3 and 50 U/ml penicillin/streptomycin, and the cells were released by gentle trituration through decreasing bore Pasteur pipettes with fire-polished tips. The cells were plated onto culture plates with confluent, quiescent glia and cultured overnight at 37°C.

Ionic currents from trigeminal neurons were recorded in the whole-cell configuration of the patch-clamp method 43 at room temperature (22–24°C), as described in 44. The solutions used to record different types of current are listed in Table 1. Dishes were continually perfused with HEPES buffered saline (HBS, Solution 1). Calcium channel currents (I_Ca), were recorded in Solution 2, recordings of sodium channel currents (I_Na) were made in Solution 3. Recordings of I_Ca and I_Na were made with fire polished borosilicate pipettes (A-M Systems #603500, Carlsborg WA) filled with (in mM): CsCl 120, MgATP 5, NaCl 5, Na₂GTP 0.3, EGTA 10, CaCl₂ 2 and HEPES 20, pH 7.3, resistance approximately 2 MΩ. In the cells where I_Ca and I_Na were recorded, capsaicin currents were also recorded with the above internal solution. Action potentials were recorded in Solution 4, with an internal solution that consisted of (mM): K methanesulphonate 115, KCl 5, NaCl, 8, MgCl₂, 1, MOPS 10, MgATP 2, Na₂GTP 0.3, BAPTA-K₄ 10, pH 7.0. In these recordings thin walled 7052 type glass (Garner Glass Company, Claremont CA) was used.

Recordings were made using either an Axopatch 1D amplifier (Axon Instruments, Union City, CA, USA) or a HEKA EPC 9 amplifier using Pulse acquisition and analysis software (HEKA). Currents were filtered at 3–5 kHz, sampled at 20–100 KHz, and recorded on hard disk for later analysis. Series resistance ranged from 2–7 MOhm and was compensated by 80% in all experiments. An approximate value of whole cell capacitance was determined by nulling the amplifier capacitance compensation circuit (Axoptach 1D) or automatically by EPC 9. Leak current was subtracted on line using a P/8 protocol. Cells were exposed to drugs via a series of flow pipes positioned about 200 μM from the cells. Fast application of ATP, capsaicin and acid were made with valve controlled sewer pipes.

Significant differences between means were tested using unpaired, two tailed Students t-test as noted. All data are expressed as mean ± S.E.mean unless otherwise indicated.

DAMGO ([Tyr-D-Ala-Gly-MePhe-Gly-ol]enkephalin), U-69593 ((+)-(5-, 7-, 8-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide), deltorphin II, capsaicin, laminin, polylysine were from Sigma. Buffer salts were from Sigma. F-12, L-15 and fetal bovine serum were from GIBCO. Tetrodotoxin and human NT-3 were from Alomone Laboratories. NGF (mouse 2.5 S) was from either Upstate or Sigma. Papain and collagenase were from Worthington Biochemical Corporation (Freehold, NJ, USA), dispase was from Roche Applied Sciences.