Article Figures & Data
Figures
- Fig. 1.
Strategy to distinguish nociceptors from mechanosensors. Low-threshold (non-nociceptive) mechanosensors were isolated from the mesencephalic nucleus of the fifth nerve, which contains only muscle spindle and fine-touch receptor afferents (Cody et al., 1972) (muscle spindle shown). Tooth pulp, an organ from which pain is the only conscious sensation (Ahlquist et al., 1984; Narhi et al., 1994), was used to obtain nociceptors. A dye (DiI), placed in small cavities drilled in rat molars ∼1 week before harvesting the trigeminal ganglia, is transported to the cell body, where its fluorescence distinguishes tooth-pulp afferents from other dissociated trigeminal sensory neurons (Fig. 2).
- Fig. 2.
Opioids inhibit Ca channels on nociceptive but not on non-nociceptive sensory neurons. A, Bright-field (a) and fluorescence (b) photographs of sensory neurons dissociated from the trigeminal ganglion 1 week after DiI was placed in tooth pulp; the fluorescent cell would be taken as a tooth-pulp nociceptor. A,c, Nociceptor currents carried by 1 mmBa2+ through Ca channels activated by the indicated ramp of voltage just before (control), 20 sec after the application of 1 μm DAMGO, and 40 sec after removal (recovery). A control record in 1 mm Cd2+ (which blocks all Ca channels) was subtracted from all test records. Opioid receptor antagonists (10 μm naloxone or 200 nm CTAP) fully blocked DAMGO action (data not shown). B, a, Bright-field photograph of three neurons dissociated from the mesencephalic nucleus and placed on a bed of glial cells; all three would be accepted as low-threshold (non-nociceptive) mechanosensors. B, b, Ca channels were not inhibited by DAMGO in this or any other mesencephalic neuron. Scale bars, 30 μm.
- Fig. 3.
Use of competitive RT-PCR to quantify single-cell MOR and GPD mRNAs. A, Ethidium-stained agarose gels of PCR products for MOR (top) and GPD (bottom) for 12 individual nociceptors (lanes A–L) and seven calibration tubes (left lanes). Each gel shows the reaction products from a particular run of the PCR machine. mut and m1, Mutants of MOR and GPD DNA, respectively, having 78 and 88 bp insertions between primer sites. Twelve molecules of mut and 500 molecules of m1 were seeded into their PCR tubes. RT reactions were seeded with the indicated amount of synthetic RNA for m2, which has a 268 bp insertion into the GPD sequence. Calibration tubes, assembled and processed along with the cell tubes, contain the indicated number of w.t. MOR or GPD DNA and either 12 (MOR) or 500 (GPD) mutant molecules. MW, Molecular weight. B, Positive MOR bands were quantified (numbers below MOR gel in A) by comparison with calibration ladders and correcting for RT efficiency to obtain mRNA values. Ratios of w.t./mutant band intensities in each calibration tube are plotted against the number of initial w.t. molecules (open symbols). Polynomial equations (curves) fitted to these data are used to interpolate initial cellular w.t. content from the w.t./mutant product ratios from each cell (solid symbols).
- Fig. 4.
Most nociceptors, but only rare mechanosensors, transcribe MOR mRNA. A, MOR and GPD mRNA amplified and quantified from 15 single tooth-pulp afferents (lanes A–O). MOR mRNA expression spans from below the detection threshold to 460 molecules per cell (60 pm, assuming equal distribution throughout the spherical cell); 60% of 191 nociceptors tested had detectable MOR mRNA (Table 1). B, MOR and GPD mRNA amplified and quantified from seven individual mesencephalic neurons (lanes C–I) and three pooled samples of either five (lanes A and B) or 10 (lane J) mesencephalic neurons. Cell I is one of only two (of 72) individual mesencephalic neurons that had detectable MOR mRNA. We used the pooled samples to determine whether most mesencephalic neurons express at nonzero levels below our detection threshold or whether rare ones express high levels of MOR mRNA. Six pooled samples were prepared (4 with 5 cells in each and 2 with 10). MOR mRNA was present in only three of these pools, at levels (50, 100, and 250 molecules) similar to those in single nociceptors and similar to the two individual positive cells (lane A is a positive pool; lanes B and J are negative pools). This argues that rare cells in the mesencephalic preparation have substantial MOR mRNA, because all pools should be positive if the majority of cells express at levels just below our detection threshold.MW, Molecular weight; GPD, glycerol-3-phosphate dehydrogenase.
- Fig. 5.
Large nociceptors have a low opioid response and have low MOR mRNA levels. A, Mean ± SEM percentage of Ca-channel inhibition by 1 μm DAMGO (black columns, left axis) and the fraction (±68% confidence interval) of cells inhibited by >10% (hatched columns, right axis) in tooth-pulp afferents of different cell-body diameters. The medians were 12, 17, and 5.5% for the small (<30 μm), medium (30–40 μm), and large (>40 μm) cells, respectively. There were nonresponders and very large responders (>40% inhibition) in each bin. B,Inset, The mean number of GPD (open circles, right axis) and MOR (filled circles, left axis) mRNA molecules for small, medium, and large nociceptors. GPD mRNA systematically increases with cell size, as expected when an increasing amount of cytoplasm is harvested. In contrast, MOR mRNA drops in the largest cells after increasing with cell size between the smaller cells. B, Mean concentration (moles per volume, assuming a spherical cell) of MOR mRNA (black columns,left axis) is lowest in the largest nociceptors.Asterisks indicate a significant difference from the other two means (p < 0.05).
- Fig. 6.
Opioid sensitivity is limited when the MOR mRNA is <15 pm. A, Percentage of inhibition of Ca channels by DAMGO versus the cellular MOR mRNA concentration for 120 tooth-pulp afferents (each point from a single cell). The origin (0*), which has 51 points, denotes cells with no detectable MOR mRNA. The dashed curve is the best fit (R2 = 0.51) of a single site isotherm (K1/2 = 5.4 pm;Bmax = 35%). B, The same data expressed as means ± SEM for bins of MOR mRNA concentrations. The mean opioid response systematically rises with MOR mRNA concentration until it saturates at ≥15 pm. Thenumber of cells in each bin is indicated. Except for the 10–15 pm versus >25 pm bins, values for non-neighboring bins differed significantly (p < 0.05).
- Fig. 7.
Opioid sensitivity of most large nociceptors is RNA limited. A, The mean concentration ± SEM of MOR mRNA for different-sized tooth-pulp afferents in which it was detectable (black columns, left axis) and the fraction (±68% confidence interval) of tooth-pulp afferents with detectable MOR mRNA that expressed it at >15 pm(hatched columns, right axis) decrease with increasing cell body size. Only a small fraction of large nociceptors express MOR mRNA at levels that lead to maximal opioid response. B, Hypothesis for selective opioid suppression of second pain. If protein densities at presynaptic terminals correspond to mRNA concentrations at the cell body, MOR is absent from virtually all non-nociceptive mechanosensor terminals and is present at low levels at terminals of most myelinated nociceptors.C, In this way, second pain, which is mediated by the unmyelinated terminals rich in MOR, can be selectively suppressed by opioids.
Tables
Ca channel inhibition > 10% Detectable MOR mRNA Nociceptors 77/182 109/191 Mechanosensors 0/20 2/72 Denominators indicate the total number of neurons tested. Numerators give the number that exhibited at least 10% inhibition by 1 μm DAMGO (left column) or had a detectable product of the RT-PCR for MOR mRNA (right). Many but not all nociceptors are opioid sensitive and have MOR mRNA. Mechanosensors are opioid insensitive and, with rare exceptions, did not have detectable MOR mRNA.
