Address correspondence and reprint requests to Seiji Ito, Department of Medical Chemistry, Kansai Medical University, Moriguchi 570–8506, Osaka, Japan. E-mail: firstname.lastname@example.org
Prostaglandin F2α (PGF2α) binds to its receptor (FP) to increase the intracellular-free calcium concentration ([Ca2+]i) by coupling of FP with Gq protein. Spinal intrathecal administration of PGF2α to mouse induces touch-evoked pain (mechanical allodynia), in which capsaicin-insensitive primary afferent Aβ-fibres and N-methyl-d-aspartate receptor ɛ4 subunit are involved. FP in the spinal cord, however, was not well characterized. Here, we showed constitutive expression of FP mRNA in mouse spinal cord, and functionally characterized spinal FP-expressing cells which were involved in PGF2α-induced mechanical allodynia. The method for repetitive administration of oligodeoxyribonucleotides through tubing to conscious mice was established for mechanical allodynia evaluation. We identified an antisense oligodeoxyribonucleotide targeting FP mRNA, causing both disappearance of PGF2α-induced mechanical allodynia and decrease of FP mRNA. With saline-administered mice, PGF2α rapidly increased [Ca2+]i of the cells in the deeper layer of the dorsal horn. In contrast, when the FP antisense oligodeoxyribonucleotide was repeatedly administered, the population of PGF2α-responsive cells in the slices reduced, and PGF2α-induced [Ca2+]i increase of these cells diminished. These data strongly suggested that, in the dorsal horn of the spinal cord, there are the FP-expressing cells which are involved in PGF2α-induced mechanical allodynia.
PGs are also involved in inflammation, including pain, by sensitizing peripheral terminals of primary afferent nociceptors (Vane 1971). Serendipitously, we found that touch-evoked pain, mechanical allodynia, was induced by intrathecal (i.t) administration of PGF2α, as well as PGE2, to the subarachnoid space of conscious mice (Minami et al. 1992, 1994b). PGF2α induces long-lasting mechanical allodynia, but not hyperalgesia, while PGE2 induces both mechanical allodynia and hyperalgesia (Uda et al. 1990; Minami et al. 1994b). Capsaicin treatment of neonatal mice suggests that capsaicin-insensitive, primary afferent Aβ-fibres are involved in PGF2α-induced mechanical allodynia, whereas capsaicin-sensitive C-fibres are involved in PGE2-induced allodynia (Minami et al. 1999). Further studies using mice with disrupted N-methyl-d-aspartate (NMDA) receptor genes confirm that there are two pathways and mechanisms for the mechanical allodynia (Minami et al. 1994a, 1999, 2001; Ito et al. 2001). NMDA receptor ɛ4 subunit is involved in PGF2α-induced allodynia, which is insensitive to morphine; whereas NMDA receptor ɛ1 subunit is involved in PGE2-induced allodynia, which is morphine-sensitive. As PG-induced mechanical allodynia was blocked by DL-threo-β-benzyloxyaspartate (TBOA), a non-transportable blocker of the glutamate transporter (Shimamoto et al. 1998), the glutamate transporter is also essential for the induction and maintenance of mechanical allodynia (Minami et al. 2001).
Mechanical allodynia is pain generated by innocuous tactile stimuli via primary afferent sensory neurons that terminate in the dorsal horn of the spinal cord. Thus, it is highly expected that certain cells in the spinal cord express FP. However, there are difficulties in detecting FP-expressing cells by in situ hybridization due to the very low abundance of FP mRNA, and by immunohistochemistry due to the lack of a suitable anti-FP antibody. Here, we showed that FP mRNA was detected in the spinal cord by reverse transcription-polymerase chain reaction (RT-PCR) and, then, functionally characterized FP-expressing cells that were involved in PGF2α-induced mechanical allodynia in the mouse spinal cord using antisense oligodeoxyribonucleotides (hereafter, oligonucleotides). Finally, we detected PGF2α-responsive cells in spinal cord slices by measuring [Ca2+]i and demonstrated that the FP antisense oligonucleotide affected [Ca2+]i increase.
Materials and methods
Male ddY mice (4 weeks old, weighing 22 ± 2 g; Japan SLC Ltd, Shizuoka, Japan) were used for all the experiments except [Ca2+]i measurement. The animals were maintained in climate- and light-controlled rooms, and each animal was used on one occasion only. All the experiments were conducted with the approval of the animal experimentation committees of Kansai Medical University and Osaka Medical College and in accordance with the guidelines of the Ethics Committee of the International Association for the Study of Pain (Zimmermann 1983).
Chemicals and drugs
Oligofectamine reagent and Fura 2-AM were purchased from Invitrogen (Carlsbad, CA, USA) and Wako Pure Chemicals (Osaka, Japan), respectively. λ-Carrageenan, NMDA and strychnine hemisulfate were obtained from Sigma (St Louis, MO, USA). PGE2 and PGF2α were generous gifts of Ono Central Research Institute (Osaka, Japan).
RT-PCR analysis of mRNA
Mice were decapitated before and at 1, 3, 6, and 12 h after intraplantar injection of λ-carrageenan, and the lumbar spinal cord without roots and meninges was subjected to RNA isolation and cDNA synthesis, as described previously (Doi et al. 2002). The lumber spinal cords were excised from the mice after the repetitive oligonucleotide administration and the allodynia assessment, and total RNA and cDNA were prepared in the same way. The high-stringency step-down RT-PCR (Nishizawa et al. 2000) was performed with the FP primers, AF and AR (Table 1), and the primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Nishizawa et al. 2000) as a control. The PCR protocol applied was: 10 cycles of (94°C, 1 min; 72°C, 2 min); 15 cycles of (94°C, 1 min; 65°C, 2 min; 72°C, 30 s); and 20 cycles of (94°C, 1 min; 60°C, 2 min; 72°C, 30 s) for FP, or 5 cycles for GAPDH. The PCR products (318 bp for FP, and 461 bp for GAPDH) were electrophoresed on 2.5% agarose gels. Linearity of the cDNA amplification was confirmed, and the DNA sequences were verified by sequencing, as described previously (Nishizawa et al. 2000). The nucleotide sequence data will appear in the DDBJ/EMBL/GenBank databases with the accession numbers AB073965, AB073966 and AB094411.
Table 1. Oligonucleotides used in this study
Sequence (5′→ 3′)
Oligonucleotides for antisense knockdown of FP and the RT-PCR analysis are listed. AS1 to AS5 are the antisense oligonucleotides targeting FP mRNA. Se5, Ma5, Scr5 and Random oligonucleotides were negative controls to AS5. AF and AR were a pair of primers for the RT-PCR analysis of FP mRNA. The position of nucleotides is numbered from A of the translation initiation codon of mouse FP cDNA. Sequences are depicted in the 5′-to-3′direction. Letters in lower case represent phosphorothioate-blocked nucleotides. Mutated bases of Ma5 are underlined. N of Random oligonucleotides denotes the mixture of bases A, C, G and T.
Design of FP antisense oligonucleotides
The 20-mer antisense oligonucleotides AS1-AS5 that targeted the entire coding sequence of mouse FP mRNA, various negative controls (Table 1) were designed according to the method described by Brysch (1999). Briefly, many candidate sequences without problematic motifs, such as ‘CG’ and ‘GGGG’, were designed from the mouse FP cDNA sequence (accession No. D17433 and AB094411) and screened by the blast program to eliminate candidates having unexpected homology to other mRNAs in the DDBJ/EMBL/GenBank databases. Finally, selected antisense oligonucleotides AS1-AS5 were administered to mice. Similarly, mutant oligonucleotides were designed by introducing three base mismatches to the antisense AS5 sequence and screened by the homology search to select the Ma5 oligonucleotide. The scramble control Scr5 was selected by base shuffling without changing the base composition of AS5 and by the following homology search. Random oligonucleotides were a mixture of random 20-mer oligonucleotides. All the oligonucleotides blocked by three phosphorothioate bonds at each terminus were purchased from Sigma Genosys Japan (Ishikari, Japan).
Cannulation of tubing to mouse spinal subarachnoid space
For repetitive administration of oligonucleotides and drugs to mice, we modified the previous method (Yaksh and Rudy 1976), which is optimized for rats and rabbits. Under deep anaesthesia by sodium pentobarbital (50 mg/kg), intervertebral ligaments between L5 and L6 were exposed after incision of skin and muscles and pierced by a 27-gauge needle. An 85-mm Teflon tube (inner diameter, 0.1 mm; outer diameter, 0.4 mm; Eicom, Kyoto, Japan) was curved by heat at 5 mm from an end and filled with sterile saline. The shorter part of the tube was inserted rostrally to the subarachnoid space through the intervertebral opening and fixed to muscles using 3–0 silk threads. Then, skin between the ears was longitudinally incised, and the longer part of the tube was subcutaneously pull out using a 16-gauge venous cannula (Terumo, Tokyo, Japan), and the tube was attached to the skin with silk threads. The dead volume of this tubing was about 2 μL. For administration to mice, 7 μL (per mouse) of each oligonucleotide-Oligofectamine mixture or drug was injected to the opening of the tubing between the ears using a 27-gauge needle (Terumo) and a sterile glass syringe. Mice showing signs of motor disturbance or infection (less than 3% of mice operated) were omitted.
Administration of FP antisense oligonucleotides through tubing
Saline (80 μL), containing 80 pmol of an oligonucleotide and 3.2 μL of Oligofectamine reagent, was incubated at room temperature (22–25°C) for 20 min. After addition of saline to a final volume of 200 μL, this mixture (7 μL per mouse) was injected through tubing. On day 2 and 3, the same amount of the freshly prepared oligonucleotide-Oligofectamine mixture was administered once per day. Mechanical allodynia was evaluated on day 4. As controls, saline alone or Oligofectamine reagent in saline without oligonucleotides (7 μL per mouse per day) was administered to mice for 3 days. Prior to [Ca2+]i measurement on day 4, saline alone or the AS5-Oligofectamine mixture (7 μL per mouse per day) was administered for 3 days in the same fashion.
Evaluation of mechanical allodynia
On day 4, PGF2α in saline (1 μg per mouse) was administered to mice through tubing. Five minutes after PGF2α injection, mechanical allodynia was assessed 10 times every 5 min, as described previously (Minami et al. 1992, 1994a,b). The mouse flank was lightly stroked with a paintbrush, and the allodynic response was ranked as follows: 0, no response; 1, mild squeaking with attempts to move away from the stroking probe; and 2, vigorous squeaking evoked by the stroking probe, biting at the probe, or strong efforts to escape. The scores were cumulated over a 50-min period, and thus the possible maximum cumulative score is 20 per mouse. Each cumulative score was expressed as a per cent of possible maximum cumulative score. If necessary, saline or PGE2 (10 ng per mouse) was injected as negative and positive controls, respectively. After mechanical allodynia assessment, 7 μL of 33 mm strychnine hemisulphate, a glycine receptor antagonist, was injected through tubing to confirm that the cannulated tubing was open to the spinal subarachnoid space. Unless spontaneous agitation was observed within 1 min after strychnine injection, the allodynia data of the mouse were discarded. More than 75% of mice operated responded to strychnine. The spinal cord of mice responded to strychnine was subjected further to the RT-PCR analysis to estimate the FP mRNA level.
[Ca2+]i measurement of mouse spinal cord slices
The AS5 antisense oligonucleotide or saline (control) was administered to 3-week-old mice (16–19 g) through tubing once per day for 3 days, and PGF2α-induced allodynia was evaluated on day 4, as described above. The spinal cord of mice showing expected allodynia responses was quickly excised under anaesthesia by pentobarbital and immersed in ice-cold artificial cerebrospinal fluid (ACSF; 120 mm NaCl, 3.1 mm KCl, 2.0 mm CaCl2, 1.0 mm MgCl2, 1.25 mm NaH2PO4, 25 mm NaHCO3, 5.0 mm glucose, 2.0 mm sodium pyruvate, 0.5 mmmyo-inositol, and 0.02 mm ascorbic acid). ACSF used in all the procedures of this experiment was equilibrated with 95% O2 and 5% CO2. Roots and meninges were removed from the spinal cord. The lumbar cord was embedded in 3% low-melting temperature agarose in ACSF and sliced transversely with a vibrating blade microtome (Leica VT-1000S, Nussloch, Germany). The slices (350 μm in thickness) at L4–L6 levels were placed in the recording chamber, mechanically fixed using an overlaying grid of nylon threads attached to a platinum ring, and superfused by ACSF at a flow rate of 3 mL/min. The slices were loaded by 10 μm Fura 2-AM in ACSF at room temperature for 2 h. The chamber was mounted on an inverted fluorescence microscope (Olympus IX-70, Tokyo, Japan) equipped with a dichroic mirror (505 nm) and an emission filter (515–550 nm). Fura 2 was excited at 340 and 380 nm using a high-speed excitation wavelength switcher (Hamamatsu Photonics, Hamamatsu, Japan), and the fluorescence emission signal from cells was monitored by the Aquacosmos-Ratio imaging system (Hamamatsu Photonics) with a cooled charge-coupled device camera. [Ca2+]i was expressed as a ratio of fluorescence emission intensity at 340–380 nm. To the superfusing ACSF buffer in the chamber, TBOA was added to a final concentration of 10 μm, and then PGF2α was added to a final concentration of 20 μm. After washing the chamber, NMDA was added to a final concentration of 100 μm to stimulate [Ca2+]i increase as a positive control. Cells which did not respond to NMDA were omitted.
The statistical analyses were carried out by Mann–Whitney U-test for multiple comparisons in Figs 2, 3, and 4, and Student's t-test in Fig. 6.
Expression of FP mRNA in mouse spinal cord
To examine the presence of FP mRNA in mouse spinal cord, we analyzed the FP mRNA expression by the RT-PCR technique. As shown in Fig. 1, FP mRNA was detected in the spinal cord of untreated mice (0 h, lane 1). The amplification of FP cDNA in the spinal cord required a total of 45 cycles in PCR, while that of GAPDH cDNA required 30 cycles. Thus, the expression level of FP mRNA seemed to be much lower than that of GAPDH mRNA. Intraplantar injection of carrageenan induces inflammation of paws and marked hyperalgesia, as well as the increase of prostacyclin receptor and cyclo-oxygenase 2 mRNAs (Seibert et al. 1994; Hay and de Belleroche 1997; Doi et al. 2002). When carrageenan was injected in mice, there were not apparent changes in the expression level of FP mRNA for up to 6 h following the carrageenan injection, and the level of FP mRNA declined at 12 h (lanes 2–5). These results demonstrated that FP mRNA was expressed at a low level in the mouse spinal cord and the expression was not induced by carrageenan, suggesting that FP mRNA is constitutively expressed in the spinal cord.
Antisense knockdown of FP in vivo
Next, we tried to knockdown FP mRNA in the spinal cord of living conscious mice and to examine whether PGF2α-induced mechanical allodynia was suppressed. The antisense oligonucleotides targeting mouse FP mRNA, AS1 to AS5, were designed to cover the entire coding region of FP mRNA (Table 1 and Fig. 2a). The ‘CG’ and ‘GGGG’ motifs causing non-specific biological effects (Brysch 1999), which are often seen in nucleotide sequences encoding transmembrane domains, were avoided. Single-shot i.t. administration of the FP antisense oligonucleotides with Oligofectamine reagent did not give reproducible results in PGF2α-induced allodynia (data not shown). Therefore, we tried to administer the oligonucleotides to mouse spinal subarachnoid space repetitively. As the previous method (Yaksh and Rudy 1976) is optimized for drug delivery to rats and rabbits, we modified and adapted it to mice. Through the tubing cannulated to the spinal subarachnoid space, the FP antisense oligonucleotide-Oligofectamine mixture was injected once per day for 3 days, as described in Materials and methods. PGF2α for mechanical allodynia assessment was also injected through the same tubing. After the evaluation of PGF2α-induced allodynia, strychnine, an antagonist at glycine receptors, was injected through the same tubing. When spontaneous agitation was observed within 1 min after strychnine injection, it was confirmed that the cannulated tubing was open to the subarachnoid space.
By our newly established method, the mice administered by the antisense AS5 oligonucleotide gave marked decrease of allodynia scores, compared with the saline-administered mice (Fig. 2b). In contrast, the administration of AS1, AS2, or Random oligonucleotides, which were a mixture of random 20-mer oligonucleotides as a negative control, did not diminish allodynia scores significantly. As the antisense oligonucleotide inhibits translation by sequence-specific binding to mRNA and subsequent digestion of mRNA with endogenous RNase H (Brysch 1999), AS1 near the translation initiation codon is theoretically the most effective. However, AS5 close to the termination codon was experimentally the most effective on PGF2α-induced mechanical allodynia. For further analyses, AS5 was selected.
We examined whether the antisense AS5 oligonucleotide functioned specifically to PGF2α-induced mechanical allodynia. The AS5-administered mice were assessed by sequential injection of saline, PGF2α and PGE2 through tubing. As expected, mechanical allodynia did not appear for 50 min after saline injection (Fig. 3a). After PGF2α injection to the same mice, mechanical allodynia disappeared over the 50-min period, indicating that PGF2α-induced allodynia was suppressed. When PGE2 was then injected, allodynia scores increased significantly. These results clearly demonstrated that the antisense AS5 oligonucleotide suppressed specifically PGF2α-induced allodynia. In our experiments, Oligofectamine reagent, a transfection reagent for oligonucleotides, was used to improve the incorporation of oligonucleotides into spinal cord cells. To exclude the possibility that this reagent non-specifically induced mechanical allodynia or blocked PGF2α-induced allodynia, we performed the next experiment (Fig. 3b). Oligofectamine reagent in saline without oligonucleotides was prepared and administered to mice for 3 days. On day 4, saline and PGF2α were sequentially administered through tubing for mechanical allodynia assessment. When saline was injected, mechanical allodynia was not observed. After PGF2α injection to the same mice, allodynia scores increased significantly. These data indicated that Oligofectamine reagent itself did not have any non-specific effects on mechanical allodynia. Together with these data, the Oligofectamine-mediated administration of antisense AS5 oligonucleotide to mice specifically suppressed PGF2α-induced mechanical allodynia.
To ensure the specificity in the AS5 function, we administered negative controls to mice, evaluated PGF2α-induced mechanical allodynia, and estimated the FP mRNA level in the spinal cord (Fig. 4). All the negative controls used for this study had low cross-homology to mouse mRNAs except FP mRNA by searching mouse nucleotide databases of the DDBJ/EMBL/GenBank. Se5, a sense oligonucleotide to AS5, had the same sequence of FP mRNA. Unexpectedly, it suppressed PGF2α-induced allodynia, and the level of FP mRNA was significantly lower than that of the saline-administered mice. Ma5, which was one of mutant oligonucleotides having three mismatches in the AS5 sequence, was administered to mice. Ma5 also reduced the allodynia score and the FP mRNA content. Finally, the scramble Scr5 oligonucleotide was designed by base shuffling without changing the base composition of AS5, as the scramble (or nonsense) oligonucleotide is thought to be an ideal negative control (Brysch 1999). As shown in Fig. 4, Scr5 slightly reduced the allodynia score and the FP mRNA content. Random oligonucleotides, a negative control for any mRNAs, did not suppress mechanical allodynia, or lower the level of FP mRNA. Thus, Random oligonucleotides functioned as the best negative control to AS5. Together with these results, administration of the antisense AS5 oligonucleotide targeting FP mRNA through tubing suppressed specifically PGF2α-induced mechanical allodynia and decreased FP mRNA in mouse spinal cord.
Antisense effects on [Ca2+]i in mouse spinal cord cells
To investigate the distribution of FP-expressing cells in the spinal cord, we measured [Ca2+]i in situ using spinal cord slices. For control mice, saline was administered through tubing, and mechanical allodynia was evaluated. When PGF2α-induced allodynia was observed, slices at the lumbar spinal cord were prepared from the mice. When PGF2α alone was added to the superfusion buffer, only one cell showed [Ca2+]i increase among 105 cells examined. The fluorescence signal for PGF2α-stimulated [Ca2+]i increase might be weak and transient, and it was possibly masked by rapid uptake of glutamate released from synaptic terminals. To prolong the activation of the glutamate NMDA receptor by PGF2α, we added TBOA, a blocker of the glutamate transporter (Shimamoto et al. 1998), prior to PGF2α addition. In the presence of TBOA, the [Ca2+]i fluorescence of many cells in the deeper layer (lamina III and deeper) of the dorsal horn increased immediately after PGF2α addition (Figs 5 and 6a). There were very few cells showing [Ca2+]i increase in the superficial layer, i.e. laminae I and II, of the dorsal horn (data not shown). As most subunits of the Ca2+-permeable NMDA receptor are widely expressed in the spinal cord (Watanabe et al. 1992, 1994; Mori and Mishina 1995), marked [Ca2+]i increase was observed when NMDA was added as a positive control (Fig. 5b). In summary, 93 PGF2α-responsive cells were observed among 170 NMDA-responsive cells using seven spinal slices (54.7%, Fig. 6a).
Then, we administered the AS5 antisense oligonucleotide through tubing. From the mice showing disappearance of PGF2α-induced allodynia, spinal cord slices were prepared and subjected to [Ca2+]i measurement by sequential addition of TBOA, PGF2α, and then NMDA (Fig. 5c). PGF2α-stimulated [Ca2+]i increase was observed in only 37 cells among 342 NMDA-responsive cells in the deeper layer using 11 spinal slices (10.8%, Fig. 6a). Furthermore, the peak height of PGF2α-stimulated [Ca2+]i increase reduced in PGF2α-responsive cells (Fig. 5c). When the peak ratio of PGF2α-stimulated [Ca2+]i increase to NMDA-stimulated increase was calculated, the ratio using spinal cord slices from AS5-administered mice was significantly lower than that from saline-administered mice (Fig. 6b). Therefore, the antisense AS5 oligonucleotide suppressed the FP expression in the spinal cord cells both quantitatively and qualitatively. Together with these data, it is strongly suggested that the FP-expressing cells, which are involved in PGF2α-induced mechanical allodynia, are present in the deeper layer of the dorsal horn in the spinal cord.
The central role of PGF2α and FP in pain transmission has been behaviourally studied since i.t. injection of PGF2α was found to induce mechanical allodynia in conscious mice (Minami et al. 1992, 1994a,b, 1999). It is hardly possible to detect FP-expressing cells in the spinal cord, even by very sensitive in situ hybridization, due to the low level expression of FP. Here, we clearly demonstrated that FP-expressing cells were present in mouse spinal cord by RT-PCR analysis of FP mRNA (Fig. 1). Furthermore, we newly established the antisense knockdown technique for conscious mice, validity of which was evaluated by allodynia responses, mRNA levels and [Ca2+]i increase. To repeatedly administer antisense oligonucleotides with Oligofectamine reagent, we used the tubing cannulated to the mouse spinal subarachnoid space. The reproducible results were obtained from the mice operated without any detectable motor disturbances and behavioural changes, except pain responses, for several days. Using this technique, we identified the most effective AS5 among five FP antisense oligonucleotides which covered the entire coding sequence of FP mRNA and did not include the problematic ‘CG’ and ‘GGGG’ motifs. On the basis of the action that the antisense oligonucleotide inhibits mRNA translation, AS1 near the initiation codon was theoretically the most effective. The results demonstrated that AS5 close to the termination codon had marked knockdown effects on both PGF2α-induced allodynia and the FP mRNA level in the spinal FP-expressing cells (Figs 2 and 4). Effective antisense oligonucleotides (AS3, AS4 and AS5) theoretically hybridize to single-stranded regions of FP mRNA, while an ineffective AS2 hybridizes to the hairpin structure. It suggests the correlation between the antisense effect and the hybridization potentials to the mRNA single-stranded regions.
The [Ca2+]i measurement using Fura 2 with mouse spinal cord slices provided the interesting results that most PGF2α-responsive cells located in the deeper layer in the dorsal horn (Fig. 5). Moreover, the combination with our antisense knockdown of FP in mice indicated that there were PGF2α-responsive cells in the dorsal horn and that population and [Ca2+]i increase of these cells reduced by the antisense AS5 oligonucleotide. These results coincide with the reports that signals from cutaneous low-threshold mechanoreceptors, which confer touch and innocuous sensation, are transmitted by primary afferent Aβ-fibres and that Aβ-fibres terminate at laminae III to V of the grey matter (Dray et al. 1994; Woolf 1994). Our results strongly suggest that PGF2α-responsive cells are involved in PGF2α-induced mechanical allodynia.
Are all the ‘PGF2α-responsive cells’ FP-expressing cells in the spinal cord? The present study demonstrated that FP-expressing cells existed in the spinal cord (Fig. 1). PGF2α-responsive cells in the dorsal horn were functionally characterized as the cells responsive to both PGF2α and NMDA by [Ca2+]i measurement (Figs 5 and 6). PGF2α potentiates the activation of the glutamate receptors by innocuous stimuli. It is most likely that PGF2α activates an FP-expressing cell (neuron or interneuron), and glutamate released from this cell further activates a post-synaptic, NMDA receptor-expressing neuron to elevate [Ca2+]i. This agrees with the results that the fluorescence signal of [Ca2+]i increase stimulated by PGF2α alone was weak and transient and that TBOA increased PGF2α-responsive cells. It is possible that the PGF2α-responsive cells express both FP and the NMDA receptor. These hypotheses well explain PGF2α-induced allodynia involving the glutamatergic system (Minami et al. 2001). The third possibility remains that the PGF2α-responsive cell is a glial cell, as some type of astrocytes have FP (Ito et al. 1992). The NMDA receptor, however, is expressed mainly in neurons (Mori and Mishina 1995), and PGF2α-induced allodynia cannot be well explained by this hypothesis. It is crucial to clarify in which cell FP and the glutamate receptors are expressed. To answer the question, detailed studies using electrophysiological techniques and the single-cell PCR method are required.
The features of PGF2α-induced mechanical allodynia are quite similar to those of causalgia and reflex sympathetic dystrophy after peripheral nerve injury in humans (Fields 1994) and those of allodynia induced by L5–L6 nerve ligation in rats (Plummer et al. 1992). PGF2α-induced allodynia has a distinct pathway and mechanism from PGE2-induced allodynia, although the glutamatergic system and glutamate transporters mediate induction and maintenance of both mechanical allodynia (Minami et al. 1999, 2001; Ito et al. 2001). Our newly established technique for mice, namely antisense knockdown through tubing combined with allodynia evaluation and [Ca2+]i measurement, can be applied to analyze genes encoding molecules that are involved in pain transmission. The advantage of this method is that the whole neural structure and network in the spinal cord are restored. Furthermore, many knockout mice showing various phenotypes can also be analyzed. In our laboratory, genes involved in PG-induced mechanical allodynia are now under investigation using this method to distinguish PGF2α-induced allodynia from PGE2-induced allodynia. Recently, multiple different mechanisms including pre-synaptic interactions between Aβ- and C-fibres are proposed for induction of mechanical allodynia (Bennett 1994; Cervero and Laird 1996). Further analyses of spinal cord cells expressing FP and the glutamate receptor, as well as allodynia-involving molecules, will provide an insight to elucidate the mechanism of PGF2α-induced allodynia.
This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas and Scientific Research (B)(2) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; and by a grant from the Science Research Promotion Fund of the Japan Private School Promotion Foundation.