Introduction

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Potassium channels are plasma membrane proteins involved in the regulation of neuronal membrane potential, neuronal excitability and modulation of transmitter release (Rudy, 1988). Some kinds of potassium channels are opened by depolarization; outward potassium currents limit the duration of single action potentials (delayed rectifier) or set the pattern of action potential bursts (transient or A current) (Jan and Jan; 1989; Catterall; 1988). Other potassium channels are opened or closed by second messengers, such as Ca⁺⁺, ATP, inositol triphosphate and G-proteins, to mediate the action of synaptic transmitters (Ashcroft, 1989; Brown, 1990, Latorre et al., 1984). G-proteins can modulate different types of potassium channels through a direct effect on the ionic channel or by modulating, through an enzymatic step, the cytoplasmic levels of soluble second messengers, which in turn can affect the activity of ion channels (Brown, 1990; Hille, 1994). Molecular cloning has confirmed the presence of at least four subfamilies of voltage-gated potassium channel genes, homologous to the Drosophila genes Shaker, Shab, Shaw and Shal, in various mammalian species (Tempel et al., 1988; Christie et al., 1989; Stühmer et al., 1989; Chandy et al., 1990; Swanson et al., 1990; Pak et al., 1991). The mouse Kv1.1 is a Shaker-like potassium channel widely distributed in the central nervous system (Wang et al., 1994). It is endowed with delayed rectifier properties (Hopkins and Tempel, 1992), is able to modulate neuronal function (Wang et al., 1994) and underlies protein kinase A-dependent regulation when expressed in Chinese hamster ovary cells (Bosma et al., 1993).

Stimulation of opioid and GABA_B receptors provokes the opening of several potassium channels in central neurons as documented by biochemical and electrophysiological studies (Sharma et al., 1975; Brown and Birnbaumer, 1990; Gähwiler and Brown, 1985). This regulation of ionic conductance involves the activation of a pertussis toxin-sensitive G-protein (G_i/o protein) (Gähwiler and Brown, 1985; Wang and Aghajanian, 1987, Dunwiddie and Su, 1988).

Morphine and baclofen (GABA_B agonist) administration induces central analgesia that is mediated by pertussis toxin-sensitive G-proteins (Parenti et al., 1986; Galeotti et al., 1996). Therefore, agonists of receptors coupled to pertussis toxin-sensitive G-proteins, such as GABA_B and mu opioid, are able to both hyperpolarize neuronal membranes by opening potassium channels and to produce antinociception through a pertussis toxin-inhibitable mechanism. The two effects seem to be related, because potassium channel blockers (sulfonylureas, 4-aminopyridine, tetraethylammonium) antagonize the antinociception induced by agonists of these receptors (Ocana et al., 1995; Ocana and Baeyens, 1993, Raffa and Martinez, 1995). However, neither 4-aminopyridine nor tetraethylammonium are specific blockers of a particular type of potassium channel (Cook and Quast, 1990; Halliwell, 1990). Therefore, we thought it was worthwhile to further investigate the role of potassium channels as an intracellular effector in morphine and baclofen enhancement of the pain threshold by use of a more selective potassium channel blocker. This study has been carried out with the use of aODNs, short synthetic DNA segments complementary to sequences of an mRNA target. By forming DNA/mRNA heteroduplexes, aODNs can transiently inactivate single genes, and therefore, are used both for studies of gene expression and as potential informational drugs. The effects of mKv1.1 potassium channel gene inactivation by an aODN on morphine and baclofen antinociception were evaluated in mice. The aODN was conjugated to an artificial cationic lipid (DOTAP) used as the vehicle and was administered by i.c.v. injection. Vehicle and degenerated ODN were used as controls.

	Methods

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Animals

Male Swiss albino mice (24-26 g) from Morini (San Polo d'Enza, Italy) were used. Fifteen mice were housed per cage. The cages were placed in the experimental room 24 h before the test for acclimatization. The animals were fed a standard laboratory diet and tap water ad libitum and kept at 23 ± 1°C with a 12 h light/dark cycle, light at 7 A.M.

Antisense Oligonucleotides

Phosphodiester oligonucleotides (ODNs) protected from terminal phosphorothioate double substitution (capped ODNs) against possible exonuclease-mediated degradation were purchased by Genosys (Cambridge, England) and purified by high-performance liquid chromatography. The 24-mer antisense ODN 5-d CGA CAT CAC CGT CAT GAT GAA d AGC-3 (phosphorothioate residues are underlined) complementary mKv1.1 mRNA and the 24-mer fully degenerated ODN (fdODN) 5-d NNN NNN NNN NNN NNN NNN NNN d NNN-3 (where N is G, or C, or A, or T, and phosphorothioate residues are underlined) were vehiculated intracellularly by an artificial cationic lipid (DOTAP, Boehringer-Mannheim, Mannheim, Germany) to enhance both uptake and stability, as described previously (Capaccioli et al., 1993; Quattrone et al., 1994b). aODN or degenerated ODN (100-600 µM) were preincubated at 37°C for 30 min with 13 µM DOTAP, sterilized through a 0.2-µm filter and supplied to mice by i.c.v. injection of 5 µl solution as described in the next section.

Intracerebroventricular Injection of Oligonucleotides

Mice were randomly assigned to anti-mKv1.1 aODN, degenerated ODN, vehicle, saline or naive group. The antisense and degenerated ODNs were dissolved in a vehicle constituted by DOTAP. Each group received a single i.c.v. injection on days 1, 4 and 7, whereas naive animals did not receive any pretreatment.

Intracerebroventricular administration was performed under ether anesthesia with isotonic saline as a solvent, according to the method described by Haley and McCormick (1957). During anesthesia, mice were grasped firmly by the loose skin behind the head. A hypodermic needle (0.4-mm external diameter) attached to a 10-µl syringe was inserted perpendicularly through the skull and no more than 2 mm into the brain of the mouse, where 5 µl ODNs were then administered. The injection site was 1 mm to the right or left from the midpoint on a line drawn through to the anterior base of the ears. Injections were performed randomly into the right or left ventricle. To ascertain that ODNs were administered exactly into the cerebral ventricle, some mice were injected with 5 µl of diluited 1:10 Indian ink, and their brains were examined macroscopically after sectioning.

In Vitro Assays: RT-PCR-Based Analysis of mKv1.1 mRNA

Forty-eight and seventy-two hours after the last i.c.v. injection of vehicle, aODN or degenerated ODN, some mice were sacrificed and their brains were rapidly removed and stored (80°C). Mouse brain levels of Kv1.1 mRNA were determined by a quantitative RT-PCR method optimized for other genes (Quattrone et al., 1994a,1995a). Frozen mouse brain samples (0.2-0.3 g wet wt.) were homogenized in 3 volumes of RNAzol to extract total RNA according to the manufacturer's instructions. RNA was treated with RQ1 RNase-free DNase, purified by ethanol precipitation, dissolved in water containing an RNase inhibitor (RNasin at 1 U/ml) and reversely transcribed to cDNA with random hexamers. A qualitative PCR reaction was preliminarily carried out for 30 cycles in a standard hot-start PCR procedure (Chou et al., 1992) with the primer pairs A (5-GCT CTC TCC TGG CCT CCT-3; residues 544-561) and B (5-GCC CGA GAA GCT TTG-3; residues 715-732) according to the mKv1.1 cDNA published sequence (Chandy et al., 1990). Primers A and B were upstream and downstream, respectively, from the segment of mKv1.1 cDNA targeted by the anti-mKv1.1 aODN. Control PCRs were carried out on RNase treated before RT or reverse transcriptase-omitted samples to exclude any possible contamination by endogenous genomic DNA and/or by amplified DNA carry-over. Primers C (5-GCG GGA AAT CGT GCG TGA CAT-3; residues 2104-2125) and D (5-GAT GGA GTT GAA GGT AGT TTC GTG-3; residues 2409-2432), according to the published sequence (Ng et al., 1985), were used in the quantitative protocol for the amplification of -actin cDNA as an internal standard (Quattrone et al., 1995b). For both primer pairs, the three-step PCR cycles consisted of 1-min denaturation at 92°C, 1-min annealing at 56°C and 1-min extension at 72°C. PCR products were electrophoresed on 2% agarose gel and the mKv1.1 product was first identified by sequencing with the fmol sequencing kit (Promega, Madison, WI). For quantitative analysis, different volumes of mKv1.1 or -actin cDNA were separately amplified for 30 cycles of PCR, and the resulting agarose bands were analyzed by densitometry. Evaluation of mKv1.1 mRNA levels came by referring three densitometric values in the linear range obtained with the mKv1.1 PCR products to those obtained with the standard -actin PCR products, and normalizing for the relevant cDNA volumes.

Behavioral Tests

Hot-plate test. The method adopted was described by O'Callaghan and Holtzman (1975). Mice were placed inside a stainless steel container, which was set thermostatically at 52.5 ± 0.1°C in a precision water bath from KW Mechanical Workshop, Siena, Italy. Reaction times (s) were measured with a stopwatch before and 15, 30, 45 and 60 min after treatment. The endpoint used was the licking of the fore or hind paws. Those mice scoring less than 12 and more than 18 s in the pretest were rejected (30%). An arbitrary cut-off time of 45 s was adopted. Following the above-mentioned pretreatment schedule with saline, vehicle, aODN or degenerated ODN, the antinociceptive effect of morphine and baclofen was tested 72 h and 7 days after the last i.c.v. injection.

Hole-board test. The hole-board test consists in a 40-cm-square plane with 16 flush mounted cylindrical holes (diameter, 3 cm) distributed 4 by 4 in an equidistant, grid-like manner. Mice were placed on the center of the board one by one and left to move about freely for a period of 5 min each. Two electric eyes, crossing the plane from midpoint to midpoint of opposite sides, thus dividing the plane into four equal quadrants, automatically signaled the movement of the animals on the surface of the plane. Miniature photoelectric cells, in each of the 16 holes, recorded the exploration of the holes (head plunging activity) by the mice. The test was performed 72 h after the last i.c.v. injection of aODN or degenerated ODN. Naive animals were used as non-pretreated controls.

Rota-rod test. The apparatus consisted of a base platform and a rotating rod of 3-cm diameter with a nonslippery surface. The rod was placed at a height of 15 cm from the base. The rod, 30 cm in length, was divided into five equal sections by six disks. Thus up to five mice were tested simultaneously on the apparatus, with a rod-rotating speed of 16 rpm. The integrity of motor coordination was assessed on the basis of the number of falls from the rod in 30 s according to Vaught et al. (1985). The performance time was measured before and 15, 30 and 45 min after s.c. administration of saline. The test was performed 72 h after the last i.c.v. injection with aODN or degenerated ODN. Naive animals were used as un-pretreated controls.

Reagents and Drugs

Oligonucleotides used for the antisense strategy and specific primers or hexamers used for RT-PCR analysis were from Genosys (Cambridge, England). DOTAP was from Boehringer-Mannheim (Mannheim, Germany). RNAzol was from Cynna Biotecx (Houston, TX); RQ1 RNase-free DNase, RNase ONE, RNasin, Mo-MLV reverse transcriptase and fmol sequencing kit were from Promega (Madison, WI); Taq polymerase was from Perkin-Elmer-Cetus (Emeryville). The following drugs were used: (±)-baclofen (-p-chlorophenyl GABA), morphine hydrochloride (U.S.L. 10/D, Florence, Italy) and D-amphetamine (De Angeli, Florence, Italy). Baclofen, morphine and D-amphetamine were dissolved in isotonic (NaCl 0.9%) saline solution immediately before use. Antisense and degenerated ODNs were dissolved in DOTAP at least 30 min before the injection. Drug concentrations were prepared in such a way that the necessary dose could be administered in a volume of 10 ml kg¹ by s.c. injection or 5 µl per mouse by i.c.v. injection.

Sequence and Statistical Analysis

Sequences of Kv genes are from the GenBank database. Sequence comparisons of both aODN and RT-PCR primers with the database were carried out by the FASTA program. All experimental results are given as the mean ± S.E.M. An analysis of variance ANOVA, followed by Fisher's protected least significant difference procedure for post hoc comparison, was used to verify significance between two means of behavioral results. Data were analyzed with the StatView software for the Macintosh (1992). The statistical significance of RT-PCR was obtained with the Student's t-test; P values of less than 0.05 were considered significant.

	Results

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Design of oligonucleotides. Considering that the translation start sites of mRNAs are particularly prone to aODN action (Goodchild, 1989; Stein and Cheng, 1993), we compared the sequence of this site with the murine Kv1.1 cDNA and other known potassium channel coding genes, to design an effective and specific antimouse Kv1.1 aODN. As summarized in table 1, we noted that the 24-bp segment 5-GCT TTC ATC ATG ACG GTG ATG TCG -3 (residues 575-598 of the published mouse Kv1.1 cDNA sequence; Chandy et al., 1990), has a low sequence homology even with the nearest members of the Shaker-like subfamily (54% with Kv1.2, Kv1.3, Kv1.4) and is almost totally unrelated to members of other Kv gene subfamilies. We therefore designed an aODN that is complementary to this Kv1.1 mRNA segment and is probably unable to target other mouse Kv mRNAs. Moreover, this aODN has terminal GCs at both the 5 and 3 ends, which are known for enhancing the binding affinity of aODN/mRNA heteroduplexes. Considering the described sequence-independent, non-antisense effects of ODNs (Storey et al., 1991; Gao et al., 1992; Blagosklonny and Neckers, 1994; Schick et al., 1995), we designed a fully degenerated, phosphodiester-phosphorothioate-capped ODN as the most suitable control for these potentially confusing effects. The fully degenerated 24-mer is a collection of about 3 × 10¹⁴ different molecular species, so that concentrations achieved for the nanomolar to micromolar range in in vitro antisense experiments for this degenerated control, every species, i.e., every ODN of defined sequence, is present at the site of action at a concentration less than 10¹⁸ M, which is totally insufficient to achieve any antisense, or generally sequence-dependent, cellular effect. Therefore, if ODN i.c.v. administration per se had achieved any biological response, this would have been present in degenerated ODN-treated controls.

Effect of aODN on morphine and baclofen antinociception. Mice were pretreated with a single i.c.v. injection of aODN, degenerated ODN or vehicle on days 1, 4 and 7. The effect of aODN pretreatment on morphine- and baclofen-induced antinociception was then evaluated in the mouse hot-plate test.

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View larger version (29K): [in this window] [in a new window]   Fig. 1.   Prevention of morphine (7 mg kg1 s.c.)-induced antinociception by pretreatment with an aODN on mKv1.1 gene in the mouse hot-plate test. Mice were i.c.v. injected with vehicle, aODN or degenerated ODN (dODN) at the dose of 0.5 (panel A), 1.0 (panels B and D) and 3.0 nmol per injection (panel C) on days 1, 4 and 7. The hot-plate test was performed 72 h (panels A, B and C) and 7 days (panel D) after the last i.c.v. injection. Vertical lines give S.E.M.; the number of mice comprised between 9 and 17 per group. *P < .05, **P < .01 in comparison with dODN + morphine-treated mice.

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View larger version (29K): [in this window] [in a new window]   Fig. 2.   Prevention of baclofen (4 mg kg1 s.c.)-induced antinociception by pretreatment with an aODN on the mKv1.1 gene in the mouse hot-plate test. Mice were i.c.v. injected with vehicle, aODN or degenerated ODN (dODN) at the dose of 1.0 (panel A), 2.0 (panels B and D) and 3.0 nmol per injection (panel C) on days 1, 4 and 7. The hot-plate test was performed 72 h (panels A, B and C) and 7 days (panel D) after the last i.c.v. injection. Vertical lines give S.E.M.; the number of mice comprised between 10 and 17 per group. *P < .05 in comparison with dODN + baclofen-treated mice.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of increasing concentrations of an aODN on the mKv1.1 gene on morphine (7 mg kg1 s.c., panel A)- and baclofen (4 mg kg1 s.c., panel B)-induced antinociception in the mouse hot-plate test. Mice received a single i.c.v. injection of aODN (0.5, 1.0 or 2.0 nmol per injection) on days 1, 4 and 7. The hot-plate test was performed 72 h after the last i.c.v. injection. The evaluation of the analgesic effect was carried out 30 min and 45 min after administration of morphine and baclofen, respectively. Each point represents the mean of at least nine mice.

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View larger version (27K): [in this window] [in a new window]   Fig. 4.   Time course of the antinociceptive effect of morphine (5 mg kg1 s.c., panel A; 9 mg kg1 s.c., panel B) and baclofen (2 mg kg1 s.c., panel C; 5 mg kg1 s.c., panel D) after pretreatment with an aODN to the mKv1.1 gene in the mouse hot-plate test. Mice were i.c.v. injected with vehicle, aODN or degenerated ODN (dODN) at the dose of 1.0 nmol (panels A and B) or 2 nmol (panels C and D) per injection on days 1, 4 and 7. The hot-plate test was performed 72 h after the last i.c.v. injection. Vertical lines give S.E.M.; the number of mice comprised between 9 and 12 per group. *P < .05 in comparison with dODN + morphine-treated mice,  P < .05 in comparison with dODN + baclofen-treated mice.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effect of i.c.v. pretreatment with an aODN to mKv1.1 gene on the antinociceptive effect produced by increasing concentrations of morphine (5-9 mg kg1 s.c., panel A) and baclofen (2-5 mg kg1 s.c., panel B) in the mouse hot-plate test. Mice received a single i.c.v. injection of aODN at the dose of 1.0 nmol (panel A) or 2.0 nmol (panel B) per injection on days 1, 4 and 7. The hot-plate test was performed 72 h after the last i.c.v. injection. The evaluation of the analgesic effect was carried out 30 min and 45 min after administration of morphine and baclofen, respectively. Each point represents the mean of at least nine mice.

Effect of aODN on mouse behavior. Mice pretreated with aODN (3.0 nmol per injection) or degenerated ODN (3.0 nmol per injection) were evaluated for motor coordination, spontaneous motility and inspection activity by use of the rota-rod (fig. 6, panel A) and hole board (fig. 6, panel B) tests. Both tests were performed 72 h after the last i.c.v. injection. The motor coordination of animals pretreated with aODN and degenerated ODN, which was evaluated in the rota-rod test, was not significantly impaired in comparison with non-pretreated (naive) mice as revealed by pretest values (fig. 6, panel A). The successive s.c. injections did not elicit any behavioral side effects because each group progressively reduced its number of falls (fig. 6, panel A) and because mice learned how to balance on the rotating rod.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Lack of effect of repeated injections of an aODN on mKv1.1 gene in the mouse rota-rod test (panel A) and in the mouse hole-board test (panel B) in comparison with non-pretreated (naive) mice. Mice were injected with aODN (3.0 nmol per injection) or degenerated ODN (dODN) (3.0 nmol per injection) on days 1, 4 and 7. The rota-rod and hole board tests were performed 72 h after the last i.c.v. injection. D-Amphetamine (1 mg kg1 s.c.) was used as a reference drug in the hole-board test. Vertical lines give S.E.M.; the number of mice is reported in parentheses. *P < .05 in comparison with non-pretreated (naive) mice.

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Effect of aODN on specific inhibition of mKv1.1 gene expression. The lowering of Kv1.1 mRNA after aODN administration as an index of Kv1.1 gene expression inactivation was quantified by RT-PCR. Before quantification, RT products were preliminarily tested for possible genomic DNA contamination. To this purpose, amplification products, a segment of 189 bp for mouse Kv1.1 cDNA and of 234 bp for -actin cDNA, were visualized by agarose gel electrophoresis. Gel analysis (fig. 7, panel A) showed bands of expected length (lanes 1 and 2) and an absence of any contamination in the negative controls (lane 3 and 4). Quantitative results of Kv1.1 and -actin mRNA brain levels after aODN mouse treatment confirmed that phenotypic effects of anti-Kv1.1 aODN on morphine- and baclofen-induced antinociception were actually caused by the specific inhibition of Kv1.1 gene expression. Figure 7, panel B, shows that the Kv1.1 mRNA/-actin mRNA ratio was sharply lowered in anti-mKv1.1 aODN-treated mice as compared with degenerated ODN-treated mice. This decrease was aODN dose-dependent.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   (panel A) Agarose gel electrophoresis of RT-PCR products obtained by mKv1.1 or -actin mRNA from untreated mouse brain. Lane 1, -actin cDNA amplification product (234 bp); lane 2, mKv1.1 cDNA amplification product (189 bp); lane 3, negative control (RNase-pretreated); lane 4, negative control (cDNA-omitted). The expected amplification products were obtained, no contaminations appeared in the negative control lanes. (panel B) Quantitative RT-PCR analysis of mKv1.1 mRNA. After ODN treatment, total RNA was extracted from the brains of behaviorally processed mice. mKv1.1 and -actin mRNA were submitted to RT-PCR as reported under "Methods." Amplification products were analyzed on agarose gel and quantified by densitometry. Within the linearity of PCR, the amount of mKv1.1 mRNA relative to -actin mRNA has been calculated and expressed as percentage of the untreated controls. Data are the means of three determinations.

	Discussion

Top Abstract Introduction Methods Results Discussion References

The present study provides evidence for the involvement of voltage-gated potassium channels in central antinociception induced by morphine and baclofen. Pretreatment with i.c.v. administration of an antisense to the mKv1.1 gene coding for the mouse Shaker-like potassium channel 1 inhibits morphine and baclofen analgesia. mKv1.1 is a potassium channel of the Shaker-like subfamily that, when expressed in Xenopus oocytes, gives rise to a fast activating, slowly inactivating potassium current (Hopkins and Tempel, 1992). The investigation into the involvement of mKv1.1 in central analgesia was carried out on the basis of its wide distribution in the mammalian brain including areas that are involved in the regulation of the pain threshold (Wang et al., 1994). Furthermore, in the hippocampus, the Kv1.1 protein is localized at or near synaptic zones which may be important for repolarizing the membrane locally near or at the synaptic terminal and hence regulating the duration or amount of neurotransmitter release at a specific terminal (Wang et al., 1994).

The involvement of the Kv1.1 gene in antinoception induced by morphine and baclofen has been studied by means of the aODN strategy. An aODN is a short segment of synthetic DNA having a sequence complementary to a portion of a target mRNA. aODN specifically binds to targeted mRNA, preventing translation and/or mediating mRNA cleavage by RNase H and, therefore, down-regulating the synthesis of the encoded protein. aODN targeted to specific mRNAs have been proved extensively to be useful pharmacological tools for exploring a variety of biological processes at the molecular level by turning off specific gene expression (Wahlestedt, 1994). Low cell permeability and the high degradation of natural phosphodiester oligomers are considerable drawbacks in the application of aODNs both in vitro and in vivo. To overcome these drawbacks, phosphorothioate-capped phosphorodiester oligonucleotides, a class of ODN derivatives shown to maintain more stable and effective concentrations in the brain when compared with their unmodified counterpart (Whitesell et al., 1993), were used. Furthermore, both ODN stability and cell uptake were enhanced by associating ODNs with an artificial cationic lipid (DOTAP) used as an intracellular carrier (Capaccioli et al., 1993).

Prevention of morphine- and baclofen-induced antinociception by pretreatment with the antisense ODN targeting mKv1.1 gene mRNA indicates that Shaker-like voltage-gated potassium channels play an important role in the enhancement of the pain threshold consequent to the activation of the opioid and GABAergic systems. However, the AD₅₀ value for baclofen (1.74 ± 0.19) is slightly higher than that evaluated for morphine (0.68 ± 0.11). This difference could indicate that the Kv1.1 potassium channels may be more critical in morphine-induced than in baclofen-induced analgesia. It should be noted that the enhancement of the pain threshold produced by baclofen is more prolonged than that produced by morphine; and this longer effect might, at least in part, justify the higher doses of aODN required to prevent baclofen antinociception. No previous data regarding the modulation of the mKv1.1 channel by morphine and baclofen, which explain varying sensitivity to aODN treatment, have been reported in the literature.

Potassium channel modulation seems to be an important intracellular event in the central antinociception induced by morphine and baclofen, but it underlies very complex mechanisms. In fact, i.c.v. administration of ATP-sensitive potassium channel blockers such as sulfonylureas have been reported to antagonize morphine-induced antinociception (Wild et al., 1991; Narita et al., 1992; Ocaña and Baeyens, 1993; Roane and Boyd, 1993; Welch and Dunlow, 1993), whereas ATP-sensitive potassium channel openers such as cromakalim and pinacidil enhanced morphine antinociception in mice (Ocaña et al., 1996; Vergoni et al., 1992). In contrast, the administration of tetraethylammonium and 4-aminopyridine, blockers of other types of potassium channels such as voltage- and calcium-dependent K⁺ channels (Cook and Quast, 1990; Halliwell, 1990), did not modify morphine antinociception (Ocaña et al., 1995). These results are in agreement with previous electrophysiological studies (Cherubini and North, 1985; Williams et al., 1988; North and Williams, 1985). The pharmacological sensitivity of the K⁺ channels opened by baclofen might seem to be slightly different from those opened by morphine, because only the former are antagonized by tetraethylammonium and 4-aminopyridine in electrophysiological studies (North and Williams, 1985), even if the two blockers exhibit different degrees of inactivation (Inoue et al., 1985; Stevens et al., 1985). The antinociception induced by the GABA_B agonist baclofen was antagonized by tetraethylammonium and 4-aminopyridine, but unmodified by the sulfonylurea gliquidone (Ocaña and Baeyens, 1993). It is difficult, however, to deduce from the above-mentioned literature what type of K⁺ channel may underly the analgesic effect of baclofen because neither tetraethylammonium nor 4-aminopyridine are specific blockers of a particular type of K⁺ channel (Cook and Quast, 1990; Halliwell, 1990). Furthermore, mKv1.1 is sensitive to numerous potassium channel modulators including tetraethylammonium, 4-aminopyridine and high doses of cromakalim (Robertson and Owen, 1993; Grissmer et al., 1994; Stephens et al., 1994).

Even if there is little evidence that inhibition of adenylate cyclase has any pronounced effect on potassium channel function, it has been suggested that the reduction of cyclic AMP levels produced by neurotransmitters, such as mu opiates, may be an important step in the mechanism of potassium channel activation in neurons (Andrade and Aghajanian, 1985). Physiological evidence suggests that voltage-gated potassium channels are also modulated by hormones and neurotransmitters in many tissues. Indeed, protein kinase A modulation of delayed rectifier-type potassium currents has been shown in the heart and in lymphocytes (Giles et al., 1989; Soliven and Nelson, 1900). More recently, an increase in mKv1.1 potassium channel expression at the levels of RNA, protein and current density has been obtained in Chinese hamster ovary cells by reducing basal protein kinase A activity (Bosma et al., 1993). Because morphine and baclofen central antinociception is mediated by a G_i/o protein (Parenti et al., 1986; Galeotti et al., 1996), which in turn can inhibit adenylate cyclase activity (Brown and Birnbaumer, 1990; Hepler and Gilman, 1992), Kv1.1 potassium channels could be involved in morphine- and baclofen-induced enhancement of the pain threshold as an intracellular effector subsequent to the activation of a G_i/o protein.

The inhibition of morphine- and baclofen-induced enhancement of the pain threshold disappeared 7 days after the last i.c.v. injection of the aODN. This return of sensitivity implies both the total reversal of aODN-induced specific inhibition of mKv1.1 gene expression and a lack of damage or toxicity associated with aODN treatment. In comparison with naive and saline i.c.v. treatment, degenerated ODN and vehicle treatments did not produce any antagonism of morphine-induced antinociception, ruling out the possibility that the antagonism exerted by aODN could be caused by sequence-independent effects on cerebral structures. This claim is supported by results obtained from the quantitative RT-PCR analysis of ODN effects on mKv1.1 gene expression as compared with those on the housekeeping -actin gene. Degenerated ODN modified neither mKv1.1 nor -actin mRNA brain levels, whereas the anti-mKv1.1 aODN specifically lowered mKv1.1 mRNA brain levels in a dose dependent manner and did not affect -actin mRNA brain levels.

The range of doses of the investigated analgesic compounds used were chosen on the basis of the rota-rod test (data not shown), which was used as a behavioral control test to illustrate any side effects produced by the injection of a drug that cannot be revealed by the researcher through the observation of the animal's spontaneous behavior. Higher doses were not used because they impaired the mice's rota-rod performance by increasing the number of falls. Repetition of the test session every 15 min four times induces a progressively slight decrease in the number of falls in control animals. Therefore the lack of variation, or an increase in the number of falls after treatment, indicates an impairment of mice motor coordination that could lead to a misinterpretation of the results obtained in the analgesic test. Mice, even if still sensitive to the thermal stimulus used in the hot-plate test, may have no reaction to pain (licking of paws) because of their impaired motility. Lower doses of the analgesic drugs used could not be investigated because they were not endowed with antinociceptive properties.

In our experimental conditions, the anti-mKv1.1 aODN did not cause any detectable modification in mouse gross behavior. Moreover, mice treated with the highest dose of aODN had unimpaired motor coordination, spontaneous motility and inspection activity in comparison with degenerated ODN- and vehicle-treated groups or naive mice. The absence of pain threshold modification in aODN-treated mice excluded a direct hyperalgesic effect of aODNs. The possibility of a specific aODN effect on pain perception modulation can be ruled out, and therefore, the aODN-induced inhibition of morphine and baclofen analgesia evidenced in the hot-plate test appears highly reliable.

Seen as a whole, our data indicate that mKv1.1 potassium channel activity plays a key role in the transduction mechanism underlying central antinociception induced by morphine and baclofen, and also suggest the actual possibility of modulating potassium channel activity by the aODN strategy.