Data are expressed as the mean+S.E.M. with the number of neurons tested.
Usually one neuron was recorded in each slice and 3-4 slices were taken from one rat.
Student t test, Student pair t test, one sample t test, one way ANOVA and chi-square test were used for statistical analysis and a p value < 0.05 was considered to be significant.
Results
1. Functional heterogeneity of NOP receptors reveal by (+)-5a Compound
1.1. (+)-5a Compound activated GIRK channels in vlPAG neurons.
(+)-5a Compound (10 μM) shifted the holding current (Ihold in Fig. 3A) outwardly and increased the membrane current elicited by a hyperpolarization ramp command from -60 to -140 mV in vlPAG neurons (Fig. 3A). Fig. 3B shows the current-voltage (I-V) curves of the membrane currents in a vlPAG neuron before and after treatment with 10 μM (+)-5a Compound, and after washout. The current induced by (+)-5a Compound, obtained by subtracting the current in the control from that in the presence of (+)-5a Compound, displayed inward rectification (Fig. 3C). The mean reversal potential of (+)-5a Compound-elicited current was -90.5+1.3 mV (n=49), resembling the equilibrium potential of K+ ions estimated by the Nernst equation. Therefore, the K+ channel in vlPAG neurons activated by (+)-5a Compound, as by N/OFQ (Chiou et al., 2002a), is the GIRK channel, an inwardly rectifying K+ channel that is coupled to G proteins (Ikeda et al., 1997).
1.2. (+)-5a Compound was less potent and less efficacious than N/OFQ.
The effect of (+)-5a Compound on GIRK channels was concentration-dependent (0.1-30 μM). To establish its concentration-response curve (Fig. 4A), the magnitude of
GIRK channel activation induced by (+)-5a Compound was quantified from the percent increment of I-140 obtained in each neuron as described in the Methods and then, after normalized to the maximal increment (39%+4% increment, n=26) produced by 1 μM N/OFQ (Chiou et al., 2002a), expressed as the percentage of the maximal effect of N/OFQ. (+)-5a Compound, at 10 μM, produced a maximal effect of 47%, as compared to that generated by N/OFQ (Fig. 4A). For a comparison, the concentration-response curves of N/OFQ and Ro 64-6198, taken from our previous studies (Chiou et al., 2004;
Chiou et al., 2002a), are also shown in Fig. 4A. In this study, 0.3 μM N/OFQ was also tested and reproduced a similar increment (40.2%+5.4%, n=32) of I-140 (filled square in Fig. 4A) as it did before (37.2%+5.3%, n=17) (Chiou et al., 2002a). The estimated EC50
of (+)-5a Compound was 605+2 nM, which is about 12 times higher than that of N/OFQ, 52+6.8 nM (Chiou et al., 2002a). The concentration-response curves of (+)-5a Compound and Ro 64-6198 are similar (Fig. 4A) with Emaxs of 46.9%+4.6% and 61.5%+5.6% (Chiou et al., 2004), respectively.
1.3. (+)-5a Compound activated GIRK channels in about half of the recorded
neurons.
(+)-5a Compound (0.1-30 μM) activated GIRK channels in 49 out of 92 (53%) recorded neurons and had no effect in the remaining 43 neurons. The responsiveness (>
5% increment of I-140 and the induced current having a reversal potential around -90 mV) of a neuron was independent of its location; either at the superficial or deeper layer of the slice. The numbers of the neurons sensitive to (+)-5a Compound or Ro 64-6198 over the total numbers of tested neurons under various tested concentrations are depicted in Fig. 4A. A scatter plot for the responses of all neurons tested with (+)-5a Compound showed that there was a sharp cutoff between (+)-5a Compound-sensitive and -insensitive neurons (Fig. 4B). Even at the highest tested concentration (30 μM), (+)-5a Compound was still ineffective in some neurons (Fig. 4), as observed before with Ro 64-6198 (Chiou et al., 2004).
1.4. The effect of (+)-5a Compound was antagonized by NOP, but not opioid or M2
muscarinic, receptor antagonists.
In (+)-5a Compound-sensitive neurons, the effect of (+)-5a Compound (0.1-30 μM) in 23 out of 35 (66%) neurons recorded was antagonized by UFP-101 (Fig. 3A), which competitively antagonized the effect of N/OFQ in the same preparation (Chiou et al., 2005). However, in the remaining 12 neurons, the effects of (+)-5a Compound were not reduced by UFP-101. The effect of (+)-5a Compound was not affected by naloxone, a non-selective μ-opioid receptor antagonist. The I-140 increments after treatment with 10 μM (+)-5a Compound in the absence or presence of 1 μM naloxone were not
significantly different (119.8%+2.5% vs. 120.3%+2.4%, n=3).
Previous binding studies (Kolczewski et al., 2003) showed that (+)-5a Compound, at a higher concentration (10 μM), also had affinity at histamine H3, muscarinic and σ receptors, and sodium channels. It is unlikely that (+)-5a Compound activated GIRK channels through histamine H3 receptors since there are few histamine H3 receptors in the PAG (Pillot et al., 2002), and activation of σ receptors will block, not activate, K+ channels (Zhang and Cuevas, 2005). Among the five subtypes (M1-M5) of muscarinic receptors, the M2 subtype exists in the PAG and activation of M2 muscarinic receptors also results in GIRK channel activation (Sanada et al., 2007). Therefore, we further examined if AF-DX-116, a selective antagonist of M2 muscarinic receptors, would antagonize the effect of (+)-5a Compound. The result showed that AF-DX-116 did not alter the effect of (+)-5a Compound. After treatment with AF-DX-116 (3 μM), the I-140 increased by 10 μM of (+)-5a Compound was 99.9%+2.5% (n=6) of that before treatment.
1.5. N/OFQ activated GIRK channels via NOP receptors in (+)-5a
Compound-insensitive vlPAG neurons.
In those (+)-5a Compound-insensitive neurons, we further tested the effect of N/OFQ to verify if their NOP receptors and GIRK channels were functional. In 41 out
of 43 neurons which were insensitive to the pretreatment of (+)-5a Compound, N/OFQ (0.3 μM) activated GIRK channels (Fig. 5).in a comparable magnitude as in naïve neurons (135.1% + 3.2%, n=41 vs. 137.2%+5.3%, n=17). The effects of N/OFQ in these neurons were antagonized by UFP-101 (Fig. 5). This result suggests that (+)-5a Compound is ineffective in a subset of NOP receptors of vlPAG neurons, at which N/OFQ displays similar efficacy as in all neurons, This characteristic of (+)-5a Compound is similar to that of Ro 64-6198 (Chiou et al., 2004). Therefore, we further investigated the interactions between (+)-5a Compound and Ro 64-6198 in the same neurons.
1.6. (+)-5a Compound-insensitive neurons were also insensitive to Ro 64-6198, and
vice versa.
In a neuron that was unaffected by (+)-5a Compound, Ro 64-6198 also failed to induce any membrane current change (Fig. 6A). The same result was found in other 8 tested neurons. The I-140 values after treatment with 10 μM (+)-5a Compound were 99.7%+0.5% (n=9) of the controls and were 98.2%+1.0% (n=9) after further treatment with 10 μM Ro 64-6198. In these neurons, N/OFQ activated GIRK channels in a manner antagonized by UFP-101 (Fig. 6A-C). Conversely, in 5 neurons which were unresponsive to Ro 64-6198, further addition of (+)-5a Compound also had no effect
(Fig. 6D). The I-140 values after treatment with 10 μM Ro 64-6198 were 100.6%+0.6%
(n=5) of the controls, and were 99.6%+0.6% (n=5) after further treatment with 10 μM (+)-5a Compound. In these neurons, N/OFQ did activate GIRK channels (Fig. 6D) in a manner blocked by UFP-101 (data not shown).
1.7. (+)-5a Compound precluded the effect of Ro 64-6198.
In (+)-5a Compound-sensitive neurons, which have been treated with the maximal effective concentration of (+)-5a Compound (10 μM), further addition of Ro 64-6198 (10 μM) did not cause any additional change in membrane currents elicited by voltage ramps (Fig. 7A) in all of the 11 neurons tested. The I-140 values after treatment with 10 μM (+)-5a Compound were 118.8%+3% of the controls (n=11), and were 116.8%+4%
(n=11) after further treatment with 10 μM of Ro 64-6198. This result suggests that (+)-5a Compound precluded the effect of Ro 64-6198.
1.8. N/OFQ further enhanced GIRK current in (+)-5a Compound-sensitive
neurons.
In contrast to Ro 64-6198, N/OFQ (0.3 μM) further enhanced GIRK current in the neurons which were responsive to (+)-5a Compound (Fig. 7B). After further treatment with 0.3 μM N/OFQ, the I-140 values in the presence of the maximal effective
concentration (10 μM) of (+)-5a Compound were increased from 119.6%+3.6% to 132.6%+5.9% of the controls (n=8). The latter response is comparable to that produced by 0.3 μM N/OFQ alone (137.2%+5.3%, n=17). When N/OFQ was reduced to 50 nM, which is equi-effective to 10 μM of (+)-5a Compound, its increment on I-140 in 10 μM (+)-5a Compound-pretreated neurons was also the same as in non-pretreated neurons (129.2%+2.8%, n=4 vs. 129.6%+3.1%, n=4). This suggests that even at the maximal effective concentration, (+)-5a Compound did not interact with equi-effective concentration of N/OFQ.
1.9. Most of the (+)-5a Compound-sensitive neurons were GABAergic.
To characterize the differences between (+)-5a Compound-sensitive and -insensitive neurons, their electrophysiological properties, morphometric features and neurotransmitter identity were compared. These results are presented on Table 8 and Figure 8.
As shown on Table 8, the mean resting membrane potential, input resistance and capacitance of (+)-5a Compound-sensitive (n=78) and -insensitive (n=70) neurons were not significantly different. Morphometric analysis data showed that (+)-5a Compound-sensitive (n=8 from 6 rats) and (+)-5a Compound-insensitive (n=6 from 5 rats) neurons had comparable soma size and total dendritic length. (+)-5a
Compound-sensitive neurons had more primary dendrites (Fig. 8A), resembling the triangular and multipolar cells described in the Golgi study (Beitz and Shepard, 1985).
Besides, compared with (+)-5a Compound-insensitive neurons (Fig. 8B), the dendrites of (+)-5a Compound-sensitive neurons had more branching nodes leading to greater dendritic orders and more terminal tips. Using Sholl analysis, these two types of neurons could also be differentiated from their dendritic complexity (Fig 8C). Neurons sensitive to (+)-5a Compound had more intersections in the Sholl concentric rings particularly in the proximal regions (< 100 μm) (Fig. 8C), indicating that these neurons have more complicated dendritic arbors.
GABAergic neurons account for 50% of all the neurons in the PAG (Mugnaini and Oertel, 1985) and most of them are interneurons (Reichling and Basbaum, 1990).
We, therefore, examined whether the recorded neurons were GABAergic through a colocalization of GAD67 immmunofluorescent reactivity with Lucifer yellow, which had been filled in the recorded neuron through the recording electrode after the recording was completed. For those neurons which were sensitive to (+)-5a Compound, 31/40 (78%) were GABAergic (Fig. 9A), and the remaining 9 (22%) neurons were non-GABAergic, suggesting a higher proportion (31/40 vs. 9/40, p<0.01, chi-square test) of (+)-5a Compound-sensitive neurons are GABAergic. On the other hand, in 30 (+)-5a Compound-insensitive neurons examined, only 14 (47%) neurons were GABAergic
(Fig. 9B) and the remaining 16 (53%) neurons were non-GABAergic (Fig. 9C).
2. Quantitative study of [Tyr10]N/OFQ(1-11) in vlPAG
2.1. [Tyr10]N/OFQ(1-11) activated inwardly rectifying potassium channels.
[Tyr10]N/OFQ(1-11), at 3-300 μM, shifted the holding current (Ihold in Fig. 10A) outwardly and increased the membrane current elicited by hyperpolarization ramps from -60 to -140 mV voltage-dependently in vlPAG neurons (Fig. 10B). The currents increased at more negative potentials were greater than those at less negative potentials.
Thus, the current-voltage (I-V) relationship of [Tyr10]N/OFQ(1-11)-induced current, which was obtained by subtracting the currents in the control from that in the presence of [Tyr10]N/OFQ(1-11), is characterized with inward rectification (Fig. 10C). The reversal potential of [Tyr10]N/OFQ(1-11)-induced current was -92.4+1.8 mV (n=40), which corresponds to the equilibrium potential of potassium ions (-91 mV) according to the Nernst equation. Therefore, in vlPAG neurons, [Tyr10]N/OFQ(1-11), like N/OFQ (Liao et al., 2010), activated IRK channels which are coupled to G-protein (Ikeda et al., 1997).
2.2. [Tyr10]N/OFQ(1-11) was similarly efficacious, but less potent than, N/OFQ.
The effect of [Tyr10]N/OFQ(1-11) on GIRK channels was
concentration-dependent. To establish its concentration-response curve (Fig. 11), the magnitude of GIRK channel activation induced by [Tyr10]N/OFQ(1-11) was quantified from the increment of I-140 as described in Methods. The maximal increment was induced by 100 μM [Tyr10]N/OFQ(1-11), which was 34.9+5.5% (n=22) and similar to the maximal effect induced by N/OFQ (1 μM), being 39.4+4% (n=26), in the same preparations (Chiou et al., 2002).
In order to compare potency of [Tyr10]N/OFQ(1-11) with that of N/OFQ, Ro 64-6198 and (+)-5a Compound, the increment of [Tyr10]N/OFQ(1-11) was normalized to the maximal increment (39.4+4%) produced by 1 μM N/OFQ (Chiou et al., 2002), expressed as the percentage of the maximal effect of N/OFQ, as shown in Fig. 11. The estimated EC50 value of [Tyr10]N/OFQ(1-11) is 9.0+0.9 μM, which is about 173 times lower than that of N/OFQ, 52.0+6.8 nM (Chiou et al., 2002) obtained in the same preparations. [Tyr10]N/OFQ(1-11) is also less potent than Ro 64-6198 or (+)-5a Compound (Fig. 11)
2.3. The effect of [Tyr10]N/OFQ(1-11) was antagonized by UFP-101 but not
naloxone.
To verify if the effect of [Tyr10]N/OFQ(1-11) is mediated through NOP receptors, UFP-101 was applied after the effect of [Tyr10]N/OFQ(1-11) had reached the steady
state. UFP-101 decreased the current induced by [Tyr10]N/OFQ(1-11) but did not change its reversal potential (Fig. 10B). The I-140 induced by [Tyr10]N/OFQ(1-11) (100 μM) was reduced by UFP-101 (1 μM) from 132.1+5.9% to 118.6%+4.7% (n=7). The reversal potentials of [Tyr10]N/OFQ(1-11)-induced current in the absence and presence of UFP-101 were -92.4+1.8 mV (n=40) and -90.1+2.7 mV (n=20), respectively.
Furthermore, the effect of [Tyr10]N/OFQ(1-11) was unaffected by naloxone, a non-selective opioid receptor antagonist. The I-140 increments after treatment with 100 μM [Tyr10]N/OFQ(1-11) in the absence or presence of 1 μM naloxone were not significantly different (134.4%+5.1% vs. 134.5%+5.4%, n=6, p=0.96, one sample
t-test).
2.4. [Tyr10]N/OFQ(1-11) further enhanced GIRK current in (+)-5a
Compound-sensitive neurons.
[Tyr10]N/OFQ(1-11) (3-10 μM) activated GIRK channels in 40/60 of the recorded neurons. This phenomenon appears to be similar to the results obtained with (+)-5a Compound (Liao et al., 2010) and Ro 64-6198 (Chiou et al., 2004), which had effect on the NOP receptors in a portion of vlPAG neurons. However, the higher the concentration of [Tyr10]N/OFQ(1-11) tested, the fewer the insensitive neurons (Fig.
11A). This might due to the low potency of [Tyr10]N/OFQ(1-11). When its
concentration was too low, the GIRK current was too small to be distinguished from the baseline. This unlikes the case of (+)-5a Compound or Ro 64-6198. They were ineffective in a portion of tested neurons even at the highest tested concentrations (Chiou et al., 2004; Liao et al., 2010). A distinguished cut-off in the response histogram was observed in the neurons treated with (+)-5a Compound (Fig. 2B in Liao et al., 2010) but not that with [Tyr10]N/OFQ(1-11) (Fig. 11B).
To examine if the population of [Tyr10]N/OFQ(1-11)-sensitive vlPAG neurons are the same subset as those affected by (+)-5a Compound, we applied (+)-5a Compound first and then [Tyr10]N/OFQ(1-11) in the same vlPAG neurons. (+)-5a Compound (10 μM) activated GIRK channels in 12 out of 22 recorded neurons and had no effect in the remaining 10 neurons. In (+)-5a Compound-sensitive neurons, (+)-5a Compound reproduced an increment of I-140 (123.6%+4.2%, n=12), as did it before (118.5%+1.9%, n=26) (Liao 2010). In these (+)-5a Compound-sensitive neurons, [Tyr10]N/OFQ(1-11) (100 μM) further enhanced GIRK currents (Fig 12A), increasing I-140 to 135.6%+6.3%
of the controls (n=12), which is not different from that produced by 100 μM [Tyr10]N/OFQ(1-11) alone (134.9%+5.5%, n=22).
2.5. [Tyr10]N/OFQ(1-11) induced GIRK currents in (+)-5a Compound-insensitive
neurons.
In those (+)-5a Compound-insensitve neurons, [Tyr10]N/OFQ(1-11) was effective in activating GIRK currents in 8 out of 10 tested neurons. Fig. 12B demonstrates one of these neurons, in which (+)-5a Compound was ineffective, but [Tyr10]N/OFQ(1-11) activated GIRK channels. The increment of I-140 were 131.3%+5.9% (n=8) (Fig. 12B), which is not different from that produced by 100 μM [Tyr10]N/OFQ(1-11) alone (134.9%+5.5%, n=22).
2.6. [Tyr10]N/OFQ(1-11) precludes the effect of N/OFQ.
The interaction of [Tyr10]N/OFQ(1-11) with N/OFQ was further investigated. In neurons effectively affected by [Tyr10]N/OFQ(1-11) at the maximal effective concentration (100 μM), further addition of N/OFQ (0.3 μM) did not cause any additional change in membrane currents (Fig. 13) in all of the 7 neurons tested. The I-140
values after treatment with 100 μM [Tyr10]N/OFQ(1-11) were 137.7%+3.7% of controls (n=7), and were 138.1%+4.1% (n=7) after further treatment with 0.3 μM N/OFQ. The result suggests that [Tyr10]N/OFQ(1-11) precludes the effect (GIRK channel activation) of N/OFQ in vlPAG neurons.
3. Quantitative study of Compound 24 in vlPAG
3.1. N/OFQ activated GIRK channels in vlPAG neurons.
As reported previously, N/OFQ shifted the holding current (Ihold in Fig. 14A) of vlPAG neurons outwardly and increased the membrane current elicited by hyperpolarization ramps from -60 to -140 mV voltage-dependently (Fig. 14). The outward current induced by N/OFQ at more negative potentials was greater than that at positive potentials (Fig. 14B). Thus, the current-voltage (I-V) curve of N/OFQ-induced current, which was obtained by subtracting the control current from that in the presence of N/OFQ, was characterized with inward rectification (Fig. 14C). The reversal potential of N/OFQ-induced current was -85+8 mV (n=23), resembling the equilibrium potential of K+ ions calculated followed the Nernst equation. These results indicate that N/OFQ activated an inwardly rectifying K+ current in vlPAG neurons which is mediated by G-protein (Ikeda et al., 1997).
3.2. Compound 24 antagonized the effect of N/OFQ concentration-dependently.
Compound 24 reduced the current induced by N/OFQ but did not change its reversal potential (Fig. 14), being -85+8 mV, n=23 and -84+7 mV, n=18, respectively, before and after treatment with Compound 24. To quantitatively compare the interaction of compound 24 with N/OFQ in each neuron, N/OFQ was applied first and, then, followed by Compound 24 plus N/OFQ. Compound 24 (0.3-10 μM) concentration-dependently reduced the GIRK current induced by 0.1 μM N/OFQ (Fig.
15, filled circles). The IC50 of Compound 24 estimated from the concentration-inhibition curve was 2.6 + 0.6 μM (Fig. 15). The times to onset and steady state for the effect of Compound 24 were 10-15 and 25-30 min, respectively (Fig.
14). They are longer than those, being 6-8 and 15-20 min, respectively, needed in the same preparations for UFP-101 (Chiou et al., 2005).
3.3. Compound 24 reduced DAMGO-induced GIRK current at higher
concentrations.
Activation of MOP receptors, but not κ-opioid (KOP) or δ-opioid (DOP) receptors (Chieng and Christie, 1994), also induces GIRK current in 30-60% of vlPAG neurons recorded (Behbehani et al., 1990; Chieng and Christie, 1994; Chiou and How, 2001;
Chiou and Huang, 1999). Compound 24 was, therefore, challenged against the effect of DAMGO, a selective MOP receptor agonist, to examine the selectivity of Compound 24.
As reported previously (Chiou and How, 2001), DAMGO shifted the holding current outwardly and increased the membrane current elicited by hyperpolarization ramps (Fig.
16). The current induced by DAMGO also displayed inward rectification and its reversal potential resembles the equilibrium potential of K+ ions (Fig. 16B), indicating that DAMGO also activates GIRK channels. The effect of DAMGO was concentration-dependent but not observed in every recorded neuron (Chiou and How,
2001). Among 7 tested neurons, DAMGO (0.3 μM) activated GIRK channels in 5 neurons. In these DAMGO-sensitive neurons, further addition of 1 μM Compound 24, did not affect the GIRK current increased by DAMGO (Fig. 16A). The current after addition of 1 μM Compound 24 was 99.4+1.2 % of that with DAMGO alone. However, when the concentrations of Compound 24 were increased to 3-10 μM, it reduced the GIRK current induced by DAMGO concentration-dependently (Fig. 15, filled square).
Compound 24, apparently, was 3 folds more potent in inhibiting the effect of N/OFQ than that of DAMGO (Fig. 15).
3.4. Compound 24 had no effect on membrane current per se.
Compound 24 was tested alone to investigate whether this compound has intrinsic agonistic activity at NOP receptors of vlPAG neurons. At the concentration up to 10 μM, Compound 24 had no effect on holding current or the membrane currents elicited by hyperpolarization ramps (Fig. 17). The I-140 after treatment with Compound 24 was 99.4+1.5 (n=4) of the control. In neurons unaffected by Compound 24, baclofen, which also activates GIRK channels in vlPAG neurons (Chiou and How, 2001), did activate a K+ current characterized by inward rectification. This suggests that the negative result of Compound 24 alone is not due to a deterioration or absence of these K+ channels in the recorded neurons.
4. Quantitative study of SB-612111 in vlPAG
4.1. SB-612111 antagonized the effect of N/OFQ in a concentration-dependently
manner.
N/OFQ activated GIRK channels in vlPAG neurons (Fig. 18). The mean reversal potential of N/OFQ-induced current was -90.1+3.6 mV (n=44). To quantitatively analyze the antagonistic effect of SB-612111 against N/OFQ-induced GIRK current, 100 nM N/OFQ was applied first and, then, followed by SB-612111 plus N/OFQ.
SB-612111 (0.01-3 μM) concentration-dependently reduced the GIRK current evoked by 100 nM N/OFQ (Fig. 19, filled circles) but did not change its reversal potential (Fig.
18). The IC50 of SB-612111 estimated from the concentration-inhibition curve was 87.7+1.2 nM (Fig. 19).
4.2. SB-612111 did not affect the membrane current per se.
SB-612111 was tested alone for the possible intrinsic agonistic activity at NOP receptors of vlPAG neurons. At concentrations up to 1 μM, SB-612111 did not affect the holding current or the membrane currents elicited by voltage ramps (Fig. 20). The I-140
after treatment with SB-612111 was 101.5%+1.0% (n=7) of the control value. In these SB-612111 (1 μM) unaffected neurons, we further tested the effect of baclofen, a
GABAB receptor agonist that also activates GIRK channels in vlPAG neurons (Chiou, 2001), to verify if their GIRK channels were functional. Addition of 1 μM baclofen did activate a K+ current characterized by inward rectification (Fig. 20). This demonstrates that the negative result of SB-612111 alone is not due to an absence of GIRK channels in the recorded neurons or a deterioration of the recording condition.
4.3. SB-612111 did not affect DAMGO-induced GIRK current.
Although SB-612111 was 174-fold more selective at expressed NOP receptors than expressed μ-opioid receptors, it still has nanomolar binding affinity at μ-opioid receptors (Zaratin et al., 2004). SB-612111 was, therefore, challenged against the effect of DAMGO, a selective μ-opioid receptor agonist, to examine the selectivity of SB-612111 in PAG slices. As reported previously (Liao et al., 2009), DAMGO (0.3 μM) outwardly shifted the holding current and increased the membrane currents elicited by hyperpolarization ramps (Fig. 21). The current elicited by DAMGO was also characterized with inward rectification and had a reversal potential at -88.9+2.9 mV, resembling the equilibrium potential of K+ ions (Fig. 21C). This suggests that DAMGO also activates GIRK channels in vlPAG neurons. As reported previously (Chiou and How, 2001), DAMGO affected only a portion of vlPAG neurons. Among 13 tested neurons, DAMGO (0.3 μM) induced GIRK currents in 8 neurons. In these
DAMGO-sensitive neurons, further addition of SB-612111 at the concentration up to 1 μM, which markedly blocked the GIRK current induced by 100 nM N/OFQ (Fig. 19), did not affect the GIRK current increased by DAMGO (Fig. 21A). The current in the presence of DAMGO plus SB-612111 was 100.4+2.3 % (n=8) of that in the presence of DAMGO alone.
Discussion 1. Functional heterogeneity of NOP receptors