Nociceptin Augments K+ Currents in Hippocampal CA1 Neurons by Both ORL-1 and Opiate Receptor Mechanisms

Samuel G. Madamba, Paul Schweitzer, and George Robert Siggins

The Scripps Research Institute, Department of Neuropharmacology, La Jolla, California 92037


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Madamba, Samuel G., Paul Schweitzer, and George Robert Siggins. Nociceptin Augments K+ Currents in Hippocampal CA1 Neurons by Both ORL-1 and Opiate Receptor Mechanisms. J. Neurophysiol. 82: 1776-1785, 1999. We previously reported (see also the accompanying paper) that dynorphin A significantly enhanced the voltage-dependent K+ M-current (IM) in CA3 and CA1 hippocampal pyramidal neurons (HPNs). Because the opioid-receptor-like-1 (ORL-1) receptor shares a high sequence homology with opioid receptors and is expressed in rat hippocampus, we examined the effects of orphanin FQ or nociceptin, the endogenous ligand for the ORL-1 receptor, using the rat hippocampal slice preparation and intracellular voltage-clamp recording. Current-voltage (I-V) relationships from CA1 HPNs revealed that nociceptin superfusion induced an outward current reversing near the equilibrium potential for K+ ions. Ba2+ (2 mM) blocked this effect. The nociceptin-induced current was largest at depolarized membrane potentials, where IM is largely activated. Nociceptin concentrations of 0.5-1 µM (but not 0.1 µM) significantly increased IM relaxation amplitudes with recovery on washout. Interestingly, both the general opiate antagonist naloxone and the kappa  receptor antagonist nor-binaltorphimine (nBNI) inhibited the nociceptin-induced IM increases and outward currents in the depolarized range but not the inward current induced at hyperpolarized potentials. The putative ORL-1 receptor antagonist, [Phe1Psi (CH2-NH)Gly2]NC(1-13)NH2 (hereafter ORLAn), blocked most of the nociceptin current near rest but not the IM increase. However, ORLAn alone had direct effects similar to those of nociceptin, indicating that ORLAn might be a partial agonist. Our results suggest that nociceptin postsynaptically modulates the excitability of HPNs through ORL-1 and kappa -like opiate receptors linked to different K+ channels.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The primary structure of the recently identified heptadecapeptide called nociceptin (Meunier et al. 1995) or orphanin FQ (Reinscheid et al. 1996) (hereafter nociceptin) shares a high sequence homology with opioid peptides, and especially dynorphin A (Meunier et al. 1995). Indeed, Zhang and Yu (1995) have identified dynorphin A as a potential ligand for the opioid-receptor-like-1 (ORL-1) receptor expressed in Xenopus oocytes. This receptor also shares a high sequence homology with opiate receptors (Bunzow et al. 1994) and is localized in rat hippocampus (Anton et al. 1996; Bunzow et al. 1994). Nociceptin has been shown to hyperpolarize neurons by activating an inwardly rectifying K+ conductance in neurons of locus coeruleus (Connor et al. 1996a), dorsal raphe (Vaughan and Christie 1996), hypothalamus (Lee et al. 1997), and CA3 hippocampus (Ikeda et al. 1997). Nociceptin also inhibits Ca2+ currents in cultured hippocampal neurons (Knoflach et al. 1996), reduces field excitatory postsynaptic potentials (EPSPs) and prevents the induction of long-term potentiation (LTP) in CA1 hippocampus (Yu et al. 1997; for review see Meunier 1997).

Recent electrophysiological studies from our laboratory (Madamba et al. 1997; Moore et al. 1994; see also the accompanying paper, Madamba et al. 1999) showed that dynorphin A enhanced the noninactivating voltage-dependent K+ M-current (IM) in both CA3 and CA1 hippocampal pyramidal neurons (HPNs). This effect of dynorphin was blocked by naloxone and nor-binaltorphimine (nBNI). In most CA3 and some CA1 HPNs, the kappa  agonist U50,488h also increased IM. However, U69,593, a selective kappa 1 receptor agonist, did not increase IM relaxation amplitudes in CA1 HPNs, suggesting that a novel kappa  receptor subtype or ORL-1 might be involved. Although delta  receptor agonists decreased IM in CA3, opiate agonists selective for delta  and µ receptors had no effect on IM or other postsynaptic membrane properties in CA1.

Therefore because of the similarities between dynorphin and nociceptin, and the possibility that ORL-1 receptors might mediate the dynorphin IM effect, we examined the effects of nociceptin on CA1 neurons of the hippocampal slice preparation. Here we report that nociceptin, like dynorphin A, enhances IM by a kappa -like opiate receptor but also activates another K+ conductance via a nonopiate, putative ORL-1 receptor.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Slice preparation

We used standard intracellular voltage-clamp recording techniques in the rat hippocampal slice, prepared as described previously (Madamba et al. 1996; Schweitzer et al. 1993) and in the accompanying paper (Madamba et al. 1999). In brief, male Sprague-Dawley rats (100-170 g) were anesthetized with halothane (3%), decapitated, and their brains rapidly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) gassed with 95% O2-5% CO2. We used sharp glass micropipettes filled with 3 M KCl (tip resistance, 73 ± 3 MOmega ; mean ± SE) to penetrate CA1 neurons. Methods of superfusion, voltage-clamp recording, cell identification, drug administration, and data analysis were as described previously (Madamba et al. 1996; Schweitzer et al. 1993) and in the accompanying paper (Madamba et al. 1999).

Electrophysiological methods

Current and voltage records were acquired, stored, and analyzed as described in the accompanying paper (Madamba et al. 1999). Tetrodotoxin (TTX; 1 µM) was added to the superfusate to block Na+-dependent action potentials and inhibit synaptic transmission.

Electrophysiological protocols

CURRENT-VOLTAGE (I-V) ANALYSIS. We evaluated I-V relationships from a holding potential of -61 ± 1 mV with hyperpolarizing and depolarizing steps (1.5 s duration, 7 s apart). The I-V curves were constructed from the current values measured at the end of the voltage steps (steady-state) just before command offset, and the values obtained in control condition were subtracted from those in the presence of the tested substance to obtain the net current elicited.

M-CURRENT ANALYSIS. In CA1 HPNs, IM is commonly seen with holding potentials around -45 mV and hyperpolarizing steps of 5-25 mV and 1-s duration (Halliwell and Adams 1982; Moore et al. 1994); it then appears as a slow inward "relaxation" following the instantaneous (ohmic) inward current. We measured IM amplitude with software (Clampfit, Axon Instruments) that fitted second-order exponential curves to the IM relaxation and used the difference between the current values just after command onset and just before command offset to quantify the amplitude of IM.

Q-CURRENT ANALYSIS. We elicited the inwardly-rectifying IQ (or Ih) from a holding potential around -60 mV using a series of hyperpolarizing voltage steps in increments of 15 mV and 1.5 s duration. The IQ relaxation amplitudes were analyzed identically to those of IM.

Drug administration

Drugs and peptides were added from a stock solution to the ACSF in known concentrations immediately before administration to the slice chamber. In this study the cells were depolarized to a mean of -47 ± 0.5 mV for IM analysis but were held near resting potential between these periods (control, drug, and wash out) to avoid the instabilities that may develop with prolonged depolarization (see Halliwell and Adams 1982). For tests of the opiate antagonists, the usual protocol involved first applying the agonist (e.g., nociceptin), followed by washout with ACSF alone ("washout"), then adding naloxone or nBNI (to test their possible effects on membrane properties) followed by addition of nociceptin again but now together with the antagonist. In several neurons, we applied nBNI first (for 6-7 min), followed by addition of nociceptin in the presence of the kappa  antagonist. Similarly, in other studies we superfused the ORL-1 receptor antagonist, [Phe1Psi (CH2-NH)Gly2]NC(1-13)NH2 (ORLAn) first followed by nociceptin in the presence of the antagonist. At the end of some recordings, 2 mM Ba2+ was added to the superfusate to verify that IM relaxation currents were due to K+ currents.

We obtained nociceptin and ORLAn from Tocris Cookson (Ballwin, MO), naloxone from Sigma (St. Louis, MO), nBNI from Research Biochemicals International (Natick, MA), and TTX from Calbiochem-Novabiochem (San Diego, CA).

Quantification and statistics

All measurements are reported as means ± SE. We usually determined statistical significance by two-way ANOVA for repeated measures, followed by the Newman-Keuls post hoc test and simple main effects as warranted by significant group effects and interactions. For within-subject effects with at least 2 degrees of freedom, we used the more conservative Huynh-Feldt P value to reduce type 1 errors.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We recorded from a total of 45 CA1 HPNs. These neurons had an average resting membrane potential (RMP) of -68 ± 1 mV with a mean spike amplitude of 104 ± 3 mV (n = 28).

Nociceptin alters I-V relationships

We generated I-V relationships to study the overall effects of nociceptin on steady-state membrane properties in the depolarized and hyperpolarized ranges (Fig. 1A). Superfusion of 0.5 and 1 µM nociceptin elicited an outward current in the depolarized range that reversed at -93 mV (mean). Figure 1B shows control-subtracted graphs generated from the I-V relationships, as described in METHODS, to isolate the current elicited by 0.1, 0.5, and 1 µM nociceptin. Superfusion of 0.1 µM nociceptin did not significantly (P > 0.5) alter steady-state currents, although a slight outward current was seen at depolarized potentials (Fig. 1B, left panel; n = 4). A higher concentration of nociceptin (0.5 µM; Fig. 1, middle panel; n = 6) elicited a significant [F(7,35) = 10.118, P < 0.01] outward current at membrane potentials of -51 to -65 mV and an inward current at potentials of -121 to -135 mV, with a reversal at -94 ± 7 mV. Similarly, 1 µM nociceptin (Fig. 1, right panel; n = 6) induced a significant [F(7,35) = 43.228, P < 0.001] outward current at potentials of -47 to -61 mV, an effect reversing at -93 ± 6 mV. Although 1 µM nociceptin also induced an inward current at hyperpolarized potentials (-117 to -131 mV), this was not significantly different compared with washout (perhaps because of persistence of effect or incomplete washout).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1. Nociceptin elicits an outward current. A: selected current traces from current-voltage (I-V) curves of a representative hippocampal pyramidal neuron (HPN) held at -60 mV. The neuron was subjected to 3 different voltage steps sequentially applied and superimposed at each condition (voltage protocol, bottom left). Application of 1 µM nociceptin evoked an outward steady-state current at depolarized potentials and an inward current at the most hyperpolarized potential, with recovery on washout. Resting membrane potential (RMP) was -70 mV. B: I-V curves derived from data like that shown in A, indicating the net steady-state currents induced by nociceptin (mean data pooled from multiple neurons). Superfusion of 0.1 µM nociceptin (left panel; n = 4) did not significantly alter membrane currents, although there was a slight outward current at depolarized potentials. A higher concentration of nociceptin (0.5 µM; n = 6; middle panel) induced a significant outward current reversing at -94 mV. Similarly, 1 µM nociceptin (n = 6; right panel) induced an outward steady-state current reversing at -93 mV. In this and subsequent figures, error bars = SE.

The theoretical reversal potential for K+ calculated by the Nernst equation, in ACSF containing 3.5 mM K+ and assuming an intracellular concentration of 150 mM K+, is -98 mV. The experimental reversal potentials we obtained (-94 and -93 mV with 0.5 and 1 µM nociceptin, respectively), were thus near the theoretical reversal potential for K+ ions. To further confirm the ionic nature of the nociceptin effect, we superfused Ba2+ at a concentration of 2 mM to nonspecifically block K+ conductances. In the presence of 2 mM Ba2+, nociceptin no longer affected steady-state current recordings generated with I-V protocols (Fig. 2, A and B). Comparison of the net effect of 0.5 µM nociceptin in the absence (n = 6) or presence (n = 5) of Ba2+ (Fig. 2C) indicated that Ba2+ prevented most of the nociceptin effect, consistent with involvement of K+ conductances.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2. Barium prevents nociceptin effects. A: selected current recordings of a HPN held at -61 mV. In the presence of 2 mM Ba2+ to block K+ conductances, the application of 0.5 µM nociceptin for 5 min did not elicit an effect on this neuron. RMP in Ba2+ was -60 mV. B: steady-state currents from the neuron depicted in A: nociceptin had no effect throughout the voltage range tested when the superfusing media contained Ba2+. C: pooled data: the effect of 0.5 µM nociceptin on mean steady-state currents taken from 5-6 HPNs in the absence (open circle ; n = 6) or presence (; n = 5) of 2 mM Ba2+. The nociceptin-induced outward component was completely abolished, and only a small inward current remained.

Nociceptin augments the M-current

Superfusion of 0.5-1 µM nociceptin enhanced IM relaxation amplitudes. In the HPN recording shown in Fig. 3A, superfusion of 1 µM nociceptin enhanced IM amplitudes to 229% of control, increased input conductance and induced an outward current (391 pA) at holding potential (- - -), with recovery on washout. The average of six such experiments with 1 µM nociceptin is shown in Fig. 3D: nociceptin significantly [F(2,10) = 15.798, P < 0.01; Newman-Keuls, P < 0.01 for control and wash] enhanced mean IM amplitudes with recovery on wash out. In contrast, on average 0.1 µM nociceptin did not significantly (P > 0.5; n = 5) enhance IM amplitudes (Fig. 3B). An intermediate concentration (0.5 µM) significantly [F(2,30) = 13.832, P < 0.01; Newman-Keuls, P < 0.001 for control and wash; n = 16] increased IM relaxation amplitudes with recovery on washout (Fig. 3C). Because there are reports of ORL-1 desensitization (Connor et al. 1996a; Ma et al. 1997), in another set of five neurons we superfused 0.5 µM nociceptin twice with washout (mean 27 min) between applications. Both nociceptin applications increased IM amplitudes similarly to 263 ± 94% of control for the first and 240 ± 59% of control for the second application (measured at the 15-mV step), suggesting a minimal tachyphylaxis over this time course.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3. Nociceptin enhances IM relaxation amplitudes in HPNs. A: superfusion of 1 µM nociceptin (Noc; 4 min) increased IM (229% of control) and induced an outward holding current (391 pA), with recovery on washout (8 min). - - -, control holding current. Holding potential was -49 mV; RMP was -74 mV. B-D pooled data of the IM amplitudes averaged across multiple neurons over 5 command voltages in response to different concentrations of nociceptin (0.1, 0.5, and 1 µM). B: nociceptin 0.1 µM did not significantly (P > 0.5; n = 5) alter mean IM. C: In contrast, superfusion of 0.5 µM nociceptin (n = 16) significantly increased mean IM amplitudes with recovery on washout. D: a higher concentration of nociceptin (1 µM; n = 6) did not elicit a greater mean IM augmentation than 0.5 µM, indicating that the maximum effect was reached at 0.5 µM.

Nociceptin-induced currents have an opiate receptor-mediated component

There are conflicting reports regarding opioid receptor mediation of nociceptin effects (Connor et al. 1996a; Knoflach et al. 1996; Nicol et al. 1996; Rossi et al. 1996; Wang et al. 1996; Zhang and Yu 1995). Therefore we superfused opiate antagonists to determine whether nociceptin effects in CA1 neurons could be mediated by an opiate mechanism (Fig. 4). We first performed I-V analysis in the presence of 4 µM naloxone, a broad spectrum opiate antagonist, to investigate a possible alteration of the nociceptin-induced steady-state currents. Although naloxone had no significant effect on the I-V curve analyzed over the entire voltage range, it significantly reduced [F(1,4) = 27.14, P < 0.01] the nociceptin-induced steady-state outward current in the depolarized range (Fig. 4, B and C), where IM is largely activated. In contrast, at hyperpolarized potentials (-85 to -139 mV), naloxone did not alter the nociceptin-induced inward current (Fig. 4B).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4. Nociceptin effect partly involves an opioid receptor-mediated mechanism. A: selected current recordings from an I-V analysis. Application of 0.5 µM nociceptin (Noc; 7 min) alone evoked an outward steady-state current at depolarized potentials and an inward current at hyperpolarized potentials. After washout of nociceptin for 16 min (bringing current levels near control levels) and addition of the kappa -opioid receptor antagonist nor-binaltorphimine (nBNI; 200 µM; 11 min) to the superfusate, a 2nd application of nociceptin (0.5 µM) in the continued presence of nBNI had little effect on steady-state currents. Holding potential was -61 mV; RMP was -69 mV. B-E: pooled data from multiple HPNs. Nociceptin-induced current in normal media and in the presence of the opioid receptor antagonist naloxone (Nal). The outward component elicited by nociceptin (0.5-1 µM; open circle ) was prevented by naloxone (4 µM; black-triangle). C: the naloxone-sensitive component isolated by subtracting the curve obtained in the presence of the antagonist from the curve obtained in its absence. Naloxone abolished a voltage-dependent outward rectification, consistent with the blockade of the nociceptin-induced IM augmentation. D: like naloxone, the specific kappa -opioid receptor antagonist nBNI (200 nM; black-triangle) prevented the outward component elicited by nociceptin alone (0.5-1 µM; open circle ). E: isolation of the nBNI-sensitive component indicates that nBNI abolished a voltage-dependent outwardly rectifying component of the nociceptin effect. Data points were fitted using Origin 4.0 software (Microcal Software) with either a linear (Y = A + B * X) or a polynomial regression (A = A + B1 * X + b2 * X2) fit.

We then investigated the effect of the specific kappa -opioid receptor antagonist nBNI. As with naloxone, superfusion of 200-400 nM nBNI significantly [F(1,5) = 20.79, P < 0.01] inhibited the nociceptin-induced outward current in the depolarized range (Fig. 4, A, D, and E), while leaving the nociceptin-induced inward component unaffected. Figure 4, C and E, shows the nociceptin-induced components sensitive to naloxone and nBNI (currents elicited by nociceptin alone minus currents elicited by nociceptin in the presence of opioid antagonist). Thus isolating the naloxone- and nBNI-sensitive currents revealed two nociceptin effects: one sensitive and one insensitive to opiate antagonists.

To again ensure that desensitization to nociceptin was not a confound in these experiments, another five neurons were exposed to nociceptin only once, in the presence of nBNI. Such nBNI pretreatment significantly [ANOVA between subjects; F(1,42) = 9.075, P < 0.01; measured at -48 to -79 mV] blunted the nociceptin-induced outward steady-state current, indicating that the lack of effect of nociceptin was not due to desensitization caused by repeated applications.

Opiate antagonists block nociceptin M-current effects

Using the IM protocol, 4 µM naloxone inhibited both the nociceptin-induced IM increase and the outward holding steady-state current (Fig. 5A). Mean data pooled from a different set of five neurons (Fig. 5B) showed that nociceptin (0.5 µM, n = 3; 1 µM, n = 2) significantly [F(4,16) = 24.107, P < 0.001; Newman-Keuls, P < 0.05 for all conditions] increased IM relaxation amplitudes with recovery on washout. In the same neurons, naloxone (4 µM) alone did not significantly alter IM but prevented the nociceptin enhancement of IM amplitudes (Fig. 5B). Although naloxone alone did not affect IM, it induced a slight inward current in the depolarized range (Fig. 5C), perhaps due to block of opiate receptors activated constitutively or by endogenous opioids.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5. Naloxone blocks the nociceptin augmentation of IM. A: representative current recordings: superfusion of 0.5 µM nociceptin (Noc; 6 min) increased IM to 167% of control and induced an outward holding current (220 pA) with recovery on washout (8 min). Application of 4 µM naloxone (Nal; 10 min) did not alter IM amplitude but elicited an inward holding current. However, naloxone pretreatment blocked the effects of a 2nd application of nociceptin (Noc + Nal; 6 min). - - -, control holding current; holding potential was -48 mV; RMP was -71 mV. B: pooled data: IM relaxation amplitudes averaged over 5 voltage steps in various drug conditions, taken from 5 HPNs. Pretreatment with 4 µM naloxone blocked the nociceptin increase of IM. C: control-subtracted steady-state current showing the inward steady-state current induced by naloxone alone.

Because of the high structural homology between ORL-1 and kappa  receptors, and because nociceptin and dynorphin had similar effects on IM, we tested nBNI, the specific kappa  receptor antagonist. Superfusion of 200 nM nBNI alone did not alter IM amplitudes nor change the holding current, but blocked the nociceptin-induced IM increase and outward holding current (Fig. 6A). Subsequent application of 2 mM Ba2+ induced an inward current and totally blocked IM, verifying the involvement of K+ channels. Figure 6B shows the average of six experiments like that shown in Fig. 6A. As before, 0.5 µM nociceptin significantly [F(4,20) = 9.825, P < 0.05; Newman-Keuls, P < 0.001 for all conditions] increased mean IM amplitudes. Superfusion of 200 nM nBNI alone (n = 6) did not significantly (P > 0.05) alter IM but blocked the nociceptin-induced IM increase. In another set of five neurons, nociceptin was superfused only once to eliminate the possibility of tachyphylaxis: we pretreated slices with 200-400 nM nBNI first, followed by 0.5 µM nociceptin together with nBNI. In the presence of nBNI, 0.5 µM nociceptin did not significantly (P > 0.5) alter IM relaxation amplitudes (Fig. 6C).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6. kappa -Opioid receptor antagonist nBNI blocks the nociceptin-induced augmentation of IM. A: IM relaxations recorded from a representative HPN. Superfusion of 0.5 µM nociceptin (Noc; 8 min) elicited an outward holding current of 475 pA and increased IM to 222% of control, with recovery on washout (17 min). Superfusion of 200 nM nBNI alone for 12 min did not alter IM nor change the holding current (- - -). In the presence of nBNI, subsequent application of nociceptin (0.5 µM; 7 min) had no effect. Superfusion of 2 mM Ba2+ for 8 min totally blocked the current relaxation, indicating it was due to IM. Holding potential was -44 mV; RMP was -66 mV. B: pooled data: mean IM amplitudes over 5 hyperpolarizing steps; 0.5 µM nociceptin significantly increased IM with recovery on wash out. On average, nBNI alone (200 nM, n = 6) had no effect on IM. However, in the presence of nBNI, nociceptin (0.5 µM) did not alter IM (Noc + nBNI). C: mean IM amplitudes from a separate set of 5 HPNs. In these experiments, 200-400 nM nBNI was superfused 1st, then 0.5 µM nociceptin added together with nBNI. Once again, nociceptin in the presence of nBNI had no significant effect on IM.

ORL-1 antagonist blocks nociceptin-induced steady-state current near rest, but not IM

Recently, Guerrini et al. (1998) identified a nociceptin receptor antagonist, ORLAn. In CA1 HPNs, superfusion of 1-2 µM ORLAn alone induced steady-state currents (Fig. 7A, left panel). In the continued presence of ORLAn, 0.5 µM nociceptin had little effect near rest. For the net nociceptin-induced steady-state currents in the presence of ORLAn, we show the current induced by nociceptin alone (Fig. 7A, right panel); notice that ORLAn prevented most of the nociceptin-induced current near rest. Because ORLAn had both agonist and antagonist actions in CA1 neurons, it might be characterized as a partial agonist.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7. Putative nociceptin antagonist [Phe1Psi (CH2-NH)Gly2]NC(1-13)NH2 (ORLAn) inhibits some nociceptin effects but is a partial agonist in HPNs. A, left panel: control-subtracted plot averaged from 7 neurons showing that superfusion of 1-2 µM ORLAn alone elicited steady-state currents throughout the voltage range tested. In the presence of ORLAn, 0.5 µM nociceptin had little effect, especially near rest. Right panel: comparison of the nociceptin-induced current in the presence of ORLAn (; derived from left panel) with the nociceptin-induced current in normal media (Noc alone; taken from Fig. 2, middle panel). Nociceptin had a limited effect in the presence of ORLAn. B: ORLAn increased IM relaxation amplitudes. Superfusion of 1 µM ORLAn (6 min) elicited an outward holding current and increased IM relaxation amplitudes. In the continued presence of ORLAn, 0.5 µM nociceptin (Noc; 5 min) still caused an outward holding current and increased IM relaxation amplitude, with recovery on washout (23 min). C: IM relaxation amplitudes averaged from 7 cells. Both 1-2 µM ORLAn alone and 0.5 µM nociceptin in the presence of ORLAn increased IM relaxation amplitudes.

We also asked if ORLAn could alter IM relaxation amplitudes and block the nociceptin enhancement of IM. Interestingly, 1 µM ORLAn itself increased IM and induced an outward holding current (Fig. 7B), an effect similar to that of nociceptin. However, addition of nociceptin in the continued presence of ORLAn elicited a further enhancement of IM, suggesting lack of antagonism by ORLAn. Washout of both ORLAn and nociceptin returned IM relaxation amplitudes to control levels. A second application of 0.5 µM nociceptin again increased IM amplitudes (not shown). On average ORLAn alone and nociceptin with ORLAn both significantly [F(2,12) = 16.884, P < 0.001; Newman-Keuls; P < 0.05 for all cases] increased IM amplitudes (Fig. 7C).

In HPNs, the voltage-dependent cationic conductance termed IQ (Halliwell and Adams 1982) or Ih is seen as a slow inward current relaxation with hyperpolarizing voltage steps to potentials more negative than -65 mV. In the presence of 2 mM Ba2+ to isolate IQ, nociceptin had little effect on this current. Subsequent superfusion of 1 mM Cs+ inhibited the inward current, verifying IQ identity (Fig. 8A). In the same cells where nociceptin increased IM amplitudes (see Fig. 6B), nociceptin did not significantly (P > 0.05) alter IQ (Fig. 8B), although there was a slight increase at -123 mV (to 111% of control; n = 6).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 8. Nociceptin does not affect the hyperpolarization-activated Q-current. A: current traces from an HPN recorded in the presence of 2 mM Ba2+ to isolate IQ. Superfusion of 1 µM nociceptin did not affect the IQ relaxation amplitude. Further superfusion of 1 mM Cs+ abolished the inward relaxation, demonstrating involvement of IQ. Holding potential was -60 mV; RMP (in Ba2+) was -62 mV. B: mean IQ amplitudes of 6 HPNs exposed to 0.5 µM nociceptin (same neurons as in Fig. 6B). Although nociceptin slightly increased IQ, the augmentation was not significant (P > 0.05). The IQ relaxation was obtained by applying several hyperpolarizing steps (-15-mV increments) from a -63-mV holding potential (see METHODS).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our studies have shown that 1) nociceptin augmented Ba2+-sensitive currents that reversed near the equilibrium potential for K+; 2) naloxone and nBNI inhibited most of the nociceptin current at depolarized potentials but not that in the hyperpolarized range; 3) the ORL-1 antagonist ORLAn blocked the nociceptin currents near RMP; 4) nociceptin increased IM relaxation amplitudes; 5) naloxone and nBNI, but not ORLAn, prevented the enhancement of IM; and 6) nociceptin had little effect on IQ. These data suggest that nociceptin can postsynaptically modulate the excitability of HPNs through both ORL-1 and kappa -like opiate receptors linked to different K+ channels.

Nociceptin-evoked currents

The I-V relationships from CA1 HPNs showed that nociceptin currents reversed near -98 mV in 3.5 mM K+, suggesting that they are carried by K+ ions. Furthermore, Ba2+ inhibited the nociceptin effect, further indicating involvement of K+ conductances. Other studies have found that nociceptin hyperpolarized neurons by activating an inwardly rectifying K+ conductance in locus coeruleus (Connor et al. 1996a) and dorsal raphe (Vaughan and Christie 1996). Similarly, nociceptin hyperpolarized mouse CA3 HPNs by activating an inwardly rectifying K+ channel current coupled to a G-protein, and this effect was blocked by Ba2+ (Ikeda et al. 1997). In our hands, a low (100 nM) concentration of nociceptin had little effect on steady-state currents or IM amplitudes, whereas a slightly higher concentration (0.5 µM) elicited a marked effect throughout the voltage range tested. Superfusion of 1 µM nociceptin did not elicit an effect beyond that with 0.5 µM, indicating that the maximum effect was reached with 0.5 µM nociceptin. Therefore nociceptin has a fairly steep dose-response relationship with an apparent EC50 probably falling between 0.1 and 0.5 µM.

The nociceptin effect at depolarized membrane potentials, where IM is activated, was mostly inhibited by naloxone and nBNI. Several studies have found that naloxone did not inhibit nociceptin effects, in dorsal raphe nucleus (Vaughan and Christie 1996), Xenopus laevis oocytes coexpressing ORL-1 with potassium channel subunits (Matthes et al. 1996), and periaqueductal gray neurons (Vaughan et al. 1997). Similarly, in our study naloxone and nBNI did not alter the nociceptin current seen at hyperpolarized potentials, suggesting that a nonopioid mechanism may be involved in this voltage range (see Fig. 4).

We used a recently identified nociceptin antagonist to investigate specific ORL-1 mediated effects in CA1 HPNs. Guerrini et al. (1998) reported that ORLAn prevented the nociceptin-induced contractions of guinea pig ileum. In the presence of ORLAn, most of the nociceptin-induced steady-state current near RMP was blocked in CA1 HPNs. However, superfusion of ORLAn alone elicited an effect similar to that with nociceptin, suggesting that it may act as a partial agonist. Interestingly, Butour et al. (1998) reported that ORLAn acted as an agonist in transformed CHO cells expressing the human ORL-1 receptor. These studies indicate that ORLAn may need further characterization and also suggest possible multiple ORL-1 receptor types (see Nociceptin augments IM).

Our findings indicate that nociceptin inhibits CA1 HPNs by concomitantly activating at least two conductances: an opiate receptor-mediated outward current (most probably IM) at depolarized potentials and an ORL-1-mediated current near rest. A possible candidate for this latter ORLAn-sensitive current might be either the inward rectifier (Ikeda et al. 1997) or a voltage-independent K+ current (so-called "resting" current), that are both sensitive to Ba2+, although a more complete characterization of this current is needed. Interestingly, we reported that somatostatin also inhibits HPNs by activating both IM and a voltage-insensitive K+ current (Schweitzer et al. 1998). In rat hippocampus, IQ (or Ih) is a mixed Na+/K+ inwardly rectifying conductance activated at hyperpolarized membrane potentials. We found that nociceptin (like somatostatin) did not significantly alter IQ, indicating that this conductance is unlikely to account for the nociceptin current at hyperpolarized potentials.

Nociceptin augments IM

The nociceptin increase in IM relaxation amplitudes was blocked by naloxone and nBNI, suggesting that nociceptin may interact with a kappa  opiate receptor. Still, naloxone inhibition of nociceptin effects is controversial. The original studies that identified nociceptin/orphanin FQ (Meunier et al. 1995; Reinscheid et al. 1995) showed that nociceptin shares a high homologous sequence with dynorphin A but did not interact with the opiate receptors. However, there are some reports suggesting that the nociceptin system may interact with the opioid system. Thus Zhang and Yu (1995) reported that dynorphin inhibited forskolin-stimulated cyclic AMP through ORL-1 receptors expressed in Xenopus oocytes. Nociceptin-induced analgesia was blocked by naloxone in mice and rats (Rossi et al. 1996, 1998). In a microdialysis study of rat striatum, nociceptin-induced dopamine release was attenuated by naloxone (Konya et al. 1998). Furthermore, dynorphin A displaced the binding of nociceptin to ORL-1 receptor in the range of 10-100 nM (Meng et al. 1996), and naloxone and nBNI in the micromolar range inhibited nociceptin binding to ORL-1 in a CHO cell line expressing the human ORL-1 receptor (Fawzi et al. 1997).

We (Madamba et al. 1999, accompanying paper) have found that dynorphin A also increased IM amplitudes in CA1 HPNs, an effect also prevented by nBNI. However, the selective kappa  agonists U69,593 and U50,488h did not significantly increase IM amplitudes, suggesting that classic kappa  receptors (e.g., kappa 1) might not be involved. The receptors activated in this study could represent a different kappa  receptor subtype (e.g., kappa 2) or a splice variant not yet characterized (see Rossi et al. 1996). Another possibility is that the nociceptin (and perhaps dynorphin) enhancement of IM is mediated via a novel nociceptin receptor sensitive to opiate antagonists. In accord with this idea, ORLAn alone increased IM amplitudes but did not prevent the nociceptin enhancement of IM. Earlier studies characterizing the ORL-1 receptor have suggested splice variants that differ in conformation (Halford et al. 1995; Wang et al. 1994). Similarly, Rossi et al. (1996) have suggested that kappa 3 and/or KOR-3 receptors might be splice variants. Indeed, the kappa 3 agonist naloxone benzoylhydrazone inhibited nociceptin reduction of Ca2+ currents in rat dorsal root ganglia (Abdulla and Smith 1997). Interestingly, sigma receptors also have been implicated in some nociceptin effects (Kobayashi et al. 1997).

Several reports have suggested that ORL-1 receptors may undergo acute and homologous desensitization (Connor et al. 1996a; Ma et al. 1997). However, we did not detect desensitization of nociceptin enhancement of IM amplitudes. Furthermore, the averaged nociceptin-induced steady-state current was not significantly different between two successive applications. Similarly, the nociceptin-induced decrease of Ca2+ currents in dissociated hippocampal neurons did not desensitize after repeated applications of nociceptin (Knoflach et al. 1996).

Possible functional role for nociceptin in CA1

As noted in the preceding paper, several groups have reported that dynorphin inhibits LTP, the cellular model of learning and memory, in guinea pig hippocampus (Terman et al. 1994; Wagner et al. 1993; Weisskopf et al. 1993). Similarly, nociceptin inhibits LTP in rat CA1 hippocampus (Yu et al. 1997) and reduces voltage-sensitive calcium currents (required for CA1 LTP) in dissociated rat pyramidal neurons (Knoflach et al. 1996). In behavioral studies, both nociceptin and dynorphin (via kappa  receptors) diminish spatial learning in rats (Sandin et al. 1997, 1998). Thus endogenous nociceptin, by activating an outward current carried by two different K+ conductances, could play a role in preventing or regulating LTP induction. Conversely, dynorphin and/or nociceptin might enhance or mediate long-term depression (LTD). This possibility is supported by our recent data showing a novel form of LTD in CA1 that is blocked by naloxone (Francesconi et al. 1997). Studies in progress should help determine whether the opiate receptors and ion channels involved in this LTD are the same as those mediating dynorphin and nociceptin effects.

In an immunohistochemical study, Anton et al. (1996) found evidence for postsynaptic ORL-1 localization in the hilus of the dentate. The hilar mossy cells and somatostatin containing neurons are believed to play a role in epilepsy and seizures (for review see McNamara 1994). A recent study also showed nociceptin immunoreactivity in the hippocampus (Mitsuma et al. 1998). It is possible that nociceptin is co-localized with somatostatin or is located in hilar interneurons, where it could serve as a brake to inhibit seizures, as postulated for somatostatin (Tallent and Siggins 1997). Indeed, nociceptin inhibits epileptiform activity in CA3 neurons in slices treated with Mg2+-free ACSF (M. K. Tallent, unpublished observations). Nociceptin and somatostatin have similar actions in CA1 pyramidal neurons: augmentation of IM amplitude and induction of an outward current at rest. Somatostatin and nociceptin also both have inhibitory presynaptic actions in the hippocampus (Tallent and Siggins 1997; Yu et al. 1997).

These and other studies show that nociceptin markedly inhibits neurons by a variety of mechanisms, including activation of IM and an inwardly rectifying K+ conductance (Ikeda et al. 1997), reduction of various Ca2+ currents (Abdulla and Smith 1997; Connor et al. 1996b), and reduction of synaptic transmission (Yu et al. 1997). These effects are consistent with an anticonvulsive function. Our findings that nociceptin can augment at least two K+ conductances, one active at depolarized potentials (IM) and the other near rest and at more hyperpolarized potentials, suggest that endogenous nociceptin could provide a powerful inhibitory influence over a wide range of membrane potentials. This influence may serve to clamp the membrane potential at rest and provide an intrinsic braking mechanism against neuronal hyperexcitabilty.


    ACKNOWLEDGMENTS

We thank Drs. S. J. Henriksen and M. K. Tallent for valuable criticism.

This research was supported by National Institutes of Health Grants DA-03665, MH-44346, and K01-DA-00291.


    FOOTNOTES

Address for reprint requests: G. R. Siggins, CVN12, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 September 1998; accepted in final form 3 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society