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Journal of Physiology (2001), 532.3, pp. 701-712
© Copyright 2001 The Physiological Society
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ABSTRACT |
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(CH2-NH)Gly2]NC(1-13)NH2 (PheN/OFQ), a substance known as an antagonist/partial agonist of the ORL receptor.
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INTRODUCTION |
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Recently, a heptadecapeptide termed nociceptin (Meunier et al. 1995) or orphanin FQ (Reinscheid et al. 1995) was identified as the endogenous ligand of a G-protein-coupled opioid receptor-like (ORL) protein (Civelli et al. 1998). Despite sequence homologies with traditional opioid receptors, the ORL receptor shows low affinity binding to selective opioid agonists or antagonists. On the other hand, nociceptin/orphanin FQ (N/OFQ) does not activate classical opioid receptors (Calo' et al. 2000). Since its discovery, N/OFQ has been the subject of intensive studies dedicated to its physiological role. Perhaps the best understood function of N/OFQ is that of a pain-modulating substance (Calo' et al. 2000). In addition, N/OFQ modulates the central integration of stressful stimuli (Köster et al. 1999; Griebel et al. 1999). For instance, N/OFQ evoked anxiolytic-like effects in mice and rats, largely irrespective of the behavioural protocols that were used to generate fear-like responses by exposure to various stressful environmental conditions (Jenck et al. 1997; Griebel et al. 1999). Accordingly, genetically generated N/OFQ-deficient mice displayed elevated stress susceptibility and showed impaired adaptative responses to repeated stress (Köster et al. 1999). A central structure for the mediation of responses to emotional stress is the amygdala (Fendt & Fanselow, 1999; Maren, 1999; LeDoux, 2000). Indeed, relatively high levels of N/OFQ, its precursor protein and its receptor are found in the various subnuclei of the amygdala, as indicated by immunohistochemistry, in vitro receptor autoradiography, ligand-stimulated in situ [35S]GTP
S binding and in situ hybridization studies (Shimohira et al. 1997; Sim & Childers, 1997; Neal et al. 1999a,b). At the cellular level, application of N/OFQ induced an inwardly rectifying K+ conductance mediated by ORL receptors coupled to a pertussis toxin-sensitive G-protein in a large majority of neurons in the lateral (LA) and central amygdala (Meis & Pape, 1998). While this postsynaptic effect led to a pronounced dampening of amygdaloid cell excitability, presumably contributing to the role of N/OFQ in the reduction of fear and anxiety, the ORL protein also seems to reside on fibre processes (Anton et al. 1996). In the lateral amygdala, binding of the radioligand 125I-[14Tyr]-N/OFQ was more pronounced than ORL mRNA expression (Neal et al. 1999a), which suggests that the ORL receptors are at least in part localized at presynaptic sites. A modulation of neurotransmitter release by N/OFQ was indeed revealed in various areas of the central nervous system (Calo' et al. 2000).
Besides N/OFQ, the N/OFQ precursor protein (prepronociceptin) comprises yet another bioactive peptide, termed nocistatin. Nocistatin does not bind to the ORL receptor, but is capable of opposing N/OFQ actions in several assays in vivo and in vitro (Okuda-Ashitaka et al. 1998; Nicol et al. 1998; Hiramatsu & Inoue, 1999; Zeilhofer et al. 2000). These findings have prompted us to study the effects of N/OFQ on synaptic mechanisms in the lateral amygdala and to evaluate the possible interaction of N/OFQ with its opponent nocistatin, thereby extending our knowledge on the cellular mechanisms contributing to the influences of these neuropeptides on fear-related behaviour.
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METHODS |
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Slice preparation
After deep anaesthesia with halothane, Long Evans rats of either sex (postnatal days (P) 11-18) were decapitated as approved by the Animal Care and Use Committee. A block of brain tissue containing the amygdala was rapidly removed and placed in ice-cold oxygenated physiological saline containing (mM): KCl, 2.4; MgSO4, 10; CaCl2, 0.5; piperazine-N,N '-bis(ethanesulphonic acid) (Pipes), 20; glucose, 10; sucrose, 195 (pH 7.35). Coronal slices (300 µm thick) were cut using a vibratome (Model 1000, Ted Pella, Redding, CA, USA), and were incubated in standard artificial cerebrospinal fluid (ACSF) of the following composition (mM): NaCl, 120; KCl, 2.5; NaH2PO4, 1.25; NaHCO3, 22; MgSO4, 3; CaCl2, 1; glucose, 10; bubbled with 95 % O2-5 % CO2 to a final pH of 7.3. Slices were allowed to recover at 34 °C for 20 min, and were then maintained for up to 8 h at 24-25 °C. For recording, single slices were transferred to a submersion chamber. Slices were fixed by a silk mesh around a platinum wire and were perfused continuously at a rate of approximately 2 ml min-1 at room temperature (24-25 °C) with ACSF.
Recording techniques
Whole-cell patch-clamp recordings were performed using a patch-clamp amplifier (EPC-7, List Medical Systems, Darmstadt, Germany). Neurons were approached under visual control by differential interference contrast (Axioskop FS, Achroplan 40/w; Zeiss, Oberkochen, Germany) infrared videomicroscopy (Imago camera, TILL Photonics, Martinsried, Germany).
Patch pipettes were pulled from borosilicate glass (GC150TF-10, Clark Electromedical Instruments, Pangbourne, UK) and filled with (mM): potassium gluconate, 95; tripotassium citrate, 20; NaCl, 10; Hepes, 10; MgCl2, 1; CaCl2, 0.1; K-BAPTA, 1; MgATP, 3 (pH 7.2 with KOH) for examination of excitatory postsynaptic currents (EPSCs) and (mM): KCl, 115; NaCl, 10; Hepes, 10; MgCl2, 1; CaCl2, 0.1; K-BAPTA, 1; MgATP, 3 (pH 7.2 with KOH) for recordings of inhibitory postsynaptic currents (IPSCs). Guanosine 5' [
-thio]diphosphattrilithium salt (GDP--S, 2 mM) was included in the pipette solution for examination of spontaneous and evoked EPSCs and IPSCs, and NaGTP (0.5 mM) was added for registrations of postsynaptic effects (see Fig. 5C and D). Typical electrode resistance was 2.0-2.5 M
in the bath, with access resistance around 5 M. Errors due to series resistance were less than 2.7 mV. A liquid junction potential of 10 mV (pipette solution used for EPSC recordings) was corrected for. After obtaining the whole-cell configuration, neurons were held at -70 mV. Records were low-pass filtered at 2.5 kHz (8-pole Bessel filter).
A bipolar tungsten electrode (SNEX 200x, Science Products, Hofheim, Germany) was placed on the surface of the slice above the external capsule or within the basolateral complex as indicated in Fig. 1. EPSCs or IPSCs were elicited by stimuli of 100 µs duration, delivered by a stimulus isolator (Isoflex, AMPI, Jerusalem, Israel). Stimulus amplitude and polarity were adjusted to evoke synaptic responses 30-50 % of maximal amplitude without triggering antidromic spikes. Spontaneous miniature postsynaptic currents were recorded in the presence of 1 µM tetrodotoxin (TTX). ACSF with elevated KCl (5 mM), substituted for an equimolar amount of NaCl, was used in these experiments. EPSCs were isolated during application of 10 µM (-)-bicuculline methiodide; IPSCs were recorded in the presence of 10 µM 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(f)quinoxaline-7-sulfonamide (NBQX). N/OFQ was applied only once to each slice. Drugs were added to the external ACFS. All substances were obtained from Sigma (Diesenhofen, Germany), except for N/OFQ and nocistatin, which were purchased from Gramsch Laboratories (München, Germany) and [Phe1(CH2-NH)Gly2]NC(1-13)NH2 and NBQX, which were purchased from Tocris (Langford, UK).
Data analysis
Recordings and data analysis were performed using pClamp software operating via a Labmaster DMA interface (Axon Instruments, Foster City, CA, USA) on a PC. The peak current amplitudes of EPSCs and IPSCs were derived by averaging four consecutive responses elicited at 0.067 Hz. For determining the degree of inhibition of evoked synaptic currents by N/OFQ, amplitudes were normalized with respect to the mean value of the responses within a period of 15 min before addition of the drug. Concentration-response curves were drawn according to the equation:
y = A + {(100 - A)/[1 + (x/EC50)nH]},
where A represents the relative EPSC amplitude in the presence of saturating concentrations of N/OFQ, EC50 the half-maximal effective concentration of N/OFQ and nH the Hill coefficient. Miniature postsynaptic currents were detected by the program 'Mini-Analysis' (Jaejin software, Leonia, NJ, USA). Cumulative frequency distributions and normalized averages were calculated within a period of 5 min before and after a steady-state effect of N/OFQ had been reached. For determining the degree of inhibition of spontaneous synaptic currents by N/OFQ, frequencies or amplitudes were normalized with respect to the mean control value during the 5 min period before addition of the drug. Statistical analysis (Student's t test, Student's paired t test) was performed using Origin software (Microcal, Northampton, MA, USA). Differences were considered statistically significant at P 
0.01. Data are presented as means ± standard error of the mean (S.E.M.).
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RESULTS |
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Basic properties of evoked synaptic currents
Whole-cell patch-clamp recordings were obtained from neurons with pyramidal-like morphology in the lateral amygdala (LA), which were previously shown to possess spiny dendrites (Meis & Pape, 1998), thus most likely representing class I projection cells (McDonald, 1992). Excitatory postsynaptic currents (EPSCs) were obtained upon electrical stimulation of the external capsule (EC) during pharmacological blockade of contaminating inhibitory postsynaptic currents (IPSCs) through inclusion of 10 µM bicuculline in the bathing solution. IPSCs evoked upon stimulation within the basolateral complex were analysed in isolation after addition of the non-NMDA antagonist NBQX (10 µM). The position of the stimulating electrodes is schematically illustrated in Fig. 1.
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Figure 1. Basic properties of EPSCs and IPSCs in LA projection neurons Schematic representation of a coronal slice illustrating the location of the LA and the position of the stimulating electrodes above the external capsule (a) or the basolateral amygdaloid complex (BLA, b; modified from Paxinos & Watson, 1986). A, blockade of EPSCs (evoked by stimulation of the external capsule) with the non-NMDA receptor antagonist NBQX. B, blockade of IPSCs (elicited by intra-amygdaloid stimulation) with the GABAA antagonist bicuculline. Traces in A and B represent averages of 20 responses obtained immediately before application of the drugs and after a steady-state effect had been reached. Neurons were voltage clamped at a holding potential of -70 mV. Recordings in A were obtained in the presence of bicuculline (10 µM), recordings in B in the presence of NBQX (10 µM).
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At a holding potential of -70 mV, brief stimuli delivered to the external capsule elicited fast postsynaptic currents, which were analysed in detail in 104 neurons. NBQX (10 µM, Fig. 1A) blocked the responses to 2.4 ± 0.6 % (n = 17) of the control value, indicating mediation by glutamate receptors of the non-NMDA subtype. The mean maximal amplitude of the EPSCs was -229.2 ± 19.3 pA. The decay followed a monoexponential time course with a time constant of 7.5 ± 0.3 ms. The mean 10-90 % rise time was 2.7 ± 0.1 ms (n = 36). These kinetics are similar to those previously described by Weisskopf & LeDoux (1999) and Mahanty & Sah (1999) for EPSCs in rat LA pyramidal-like putative projection neurons.
IPSCs were measured as inward currents with Cl- as the main intracellular anionic charge carrier in 64 LA neurons. Application of the GABAA antagonist bicuculline (10 µM) reduced the maximal current amplitudes to 1.5 ± 0.6 % (n = 8) of the control value, confirming mediation by GABAA receptors (Fig. 1B). Mean maximal IPSC amplitudes were -254.5 ± 23.0 pA (n = 25). IPSCs showed a monoexponential decay with a time constant of 27.5 ± 1.5 ms, and a mean 10-90 % rise time of 3.3 ± 0.2 ms (n = 25). IPSCs displaying a slower decay compared with EPSCs have previously been described in putative projection neurons of the rat basolateral amygdala (Smith & Dudek, 1996).
Suppression of evoked EPSCs and IPSCs by N/OFQ
To avoid contamination of synaptic currents by the known postsynaptic effects of N/OFQ, the latter were blocked by inclusion of GDP--S into the pipette solution for examination of synaptic transmission (Meis & Pape, 1998). Extracellular application of N/OFQ decreased the amplitude of EPSCs in a concentration-dependent manner (Fig. 2). Representative traces recorded immediately before application of N/OFQ and after a steady-state effect had been reached are shown in Fig. 2A. For statistical evaluation, EPSC amplitudes were normalized with respect to the baseline values during the 15 min period prior to application of N/OFQ (Fig. 2B). Mean inhibition was statistically significant (P 0.01) for concentrations 
10 nM. At this concentration range, EPSC amplitudes were suppressed in 17 out of 19 cells tested. Half-maximal inhibitory suppression occurred at 21.8 ± 7.5 nM N/OFQ, with a Hill coefficient of 0.8 ± 0.2. The peptide maximally reduced EPSCs to 70.3 ± 1.7 % of the control value at a concentration of 1 µM (Fig. 2C).
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Figure 2. Suppression of evoked excitatory responses by N/OFQ A, averaged EPSCs recorded before and after addition of N/OFQ at 10 nM, 100 nM and 1 µM. Traces represent averages of 20 responses obtained immediately before application of the drugs and after a steady-state effect had been reached. B, time course of the depression of normalized EPSC amplitudes by N/OFQ at the respective concentrations. C, concentration-response relationship of the N/OFQ effect. Data are means from measurements in different numbers of cells, as indicated near the data points. The EC50 and Hill values obtained from the curve were 21.8 nM and 0.8, respectively, with a mean maximal inhibition to 70.3 % of the control value.
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To determine whether N/OFQ acts via the ORL receptor [Phe1(CH2-NH)Gly2]NC(1-13)NH2 (PheN/OFQ) was tested, which has recently been described as an ORL antagonist (Guerrini et al. 1998). Since effects as a partial agonist have also been demonstrated (for review, see Calo' et al. 2000), possible changes in EPSC amplitude caused by PheN/OFQ alone were tested first. Application of PheN/ OFQ at 1 µM had no detectable effect on excitatory synaptic transmission in all cells tested (n = 4, Fig. 3A). By comparison, PheN/OFQ prevented the suppressive action of 100 nM N/OFQ on EPSCs (n = 5, Fig. 3B), suggesting that N/OFQ exerts its action through the ORL receptor.
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Figure 3. Antagonism of N/OFQ-induced EPSC suppression by Phe A, lack of effect on EPSCs by 1 µM Phe
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The modulation of IPSCs by N/OFQ is shown in Fig. 4. Data were analysed as described for EPSCs. N/OFQ at 1 µM decreased IPSC amplitudes to 48.0 ± 6.8 % in 5 out of 8 neurons that were tested (Fig. 4B). A tenfold higher concentration of N/OFQ caused a similar reduction to 46.0 ± 5.0 % in 9 out of 10 tested cells (Fig. 4B). These results suggest that N/OFQ at 1 µM maximally depressed IPSC amplitudes, thereby resembling the concentration of N/OFQ needed to maximally affect EPSCs. In the presence of 1 µM PheN/OFQ, IPSCs were reduced by 1 µM N/OFQ to a significantly smaller extent compared with the depressant effect under control conditions (63.4 ± 6.9 %, n = 5 vs. 48.0 ± 6.8 %, n = 5), indicating mediation through ORL receptors. In two neurons, N/OFQ had no effect on IPSC amplitudes in the presence of PheN/OFQ.
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Figure 4. Suppression of evoked inhibitory responses by N/OFQ A, averaged IPSCs recorded before and after addition of N/OFQ at 1 and 10 µM. Traces represent averages of 20 responses obtained immediately before application of the drug and after a steady-state effect had been reached. B, time course of the depression of normalized IPSC amplitudes by N/OFQ at the respective concentrations.
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Effects of nocistatin on evoked EPSCs and IPSCs
Application of 10-100 µM nocistatin did not affect excitatory (n = 7, Fig. 5A) or inhibitory (n = 4, Fig. 5B) postsynaptic currents in LA cells. In addition, nocistatin did not exert direct postsynaptic effects on the recorded neurons, indicated by a lack of effect on membrane holding current or conductance (Fig. 5C). A modulatory action of nocistatin on the N/OFQ-induced G-protein-coupled K+ current (Meis & Pape, 1998) was not observed, as N/OFQ (1 µM) induced K+ currents with a similar maximal amplitude without (53.3 ± 7.3 pA, n = 8) and in the presence of nocistatin (53.0 ± 8.6 pA, n = 3).
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Figure 5. Lack of effect of nocistatin in LA neurons Normalized EPSCs (A) or IPSCs (B) before and after addition of nocistatin (10-100 µM). C and D, typical responses of LA neurons to nocistatin and N/OFQ. Note that nocistatin did not affect the membrane current or modulate the N/OFQ-induced outward current.
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Effects of N/OFQ on spontaneous miniature EPSCs
In order to investigate whether N/OFQ suppresses excitatory synaptic transmission through post- or presynaptic mechanisms, spontaneously occurring miniature EPSCs (mEPSCs) were analysed (Fig. 6). Action potential-evoked transmitter release was blocked by addition of TTX (1 µM) to the bathing solution. IPSCs were eliminated through addition of 10 µM bicuculline. Under these conditions, mEPSCs were completely blocked by 10 µM NBQX (n = 3, data not shown), indicating mediation by non-NMDA receptors. Blockade of Ca2+ influx through voltage-dependent Ca2+ channels achieved by addition of 200 µM Cd2+ had no effect on mEPSC frequency or amplitude (n = 3, data not shown). Typical examples of mEPSCs recorded under control conditions (upper traces) and in the presence of 1 µM N/OFQ (lower traces) are shown in Fig. 6A. The cumulative amplitude (Fig. 6B) and inter-event interval (Fig. 6C) frequency distributions were obtained during a 5 min period before addition of N/OFQ and after a steady-state effect had been reached (Fig. 6D). N/OFQ reduced the mean frequency of mEPSCs in 7 out of 8 cells (Fig. 6F), whereas the mean maximal amplitude remained unchanged (Fig. 6E). The mean maximal amplitude of mEPSCs was 17.6 ± 1.8 pA in the absence and 17.7 ± 1.7 pA in the presence of N/OFQ (n = 7). By contrast, the normalized mean frequency was reduced to 74.0 ± 2.6 % by N/OFQ compared with control values (n = 7, Fig. 6F). The mean frequency of mEPSCs changed significantly (P 0.01) from 2.66 ± 0.59 Hz under control conditions to 1.97 ± 0.45 Hz after addition of N/OFQ (n = 7).
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Figure 6. Effect of N/OFQ on mEPSCs A, examples of mEPSCs recorded in the presence of 1 µM TTX before (upper traces) and during action of N/OFQ (lower traces). Averaged mEPSCs are shown at an expanded time scale, as indicated. B and C, cumulative amplitude (B) and inter-event interval (C) frequency distributions obtained from the same neuron shown in A before addition of N/OFQ and after a steady-state effect had been reached. Note that N/OFQ did not affect the amplitude of mEPSCs, but shifted mEPSC inter-event intervals to larger values. D, time course of the N/OFQ effect. Only cells which were affected by N/OFQ are included. E and F, relative mEPSC amplitude (E) and frequency (F) pooled during control conditions and after addition of N/OFQ demonstrate a significant decrease in mEPSC frequency to 74.0 %, whereas the mean amplitude was left unchanged.
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Effects of N/OFQ on spontaneous miniature IPSCs
Pharmacologically isolated (1 µM TTX, 10 µM NBQX) spontaneously occurring miniature IPSCs (mIPSCs) were analysed as described before for mEPSCs (Fig. 7). The mIPSCs were completely blocked by 10 µM bicuculline (n = 3, data not shown), indicating mediation by GABAA receptors. N/OFQ affected mIPSCs in all neurons tested (n = 6). While mean maximal amplitudes of the mIPSCs were not significantly different before (49.2 ± 3.7 pA) and in the presence of N/OFQ (49.2 ± 3.6 pA, n = 6, Fig. 7B and E), the frequency of mIPSCs declined significantly (P 0.01) from 0.71 ± 0.11 to 0.6 ± 0.09 Hz after addition of N/OFQ, reflecting a reduction to 84.4 ± 1.1 % of the control value (n = 6, Fig. 7C and F).
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Figure 7. Effect of N/OFQ on mIPSCs A, examples of mIPSCs recorded in the presence of 1 µM TTX before (upper traces) and during action of N/OFQ (lower traces). Averaged mIPSCs are shown at an expanded time scale, as indicated. B and C, cumulative amplitude (B) and inter-event interval (C) frequency distributions obtained from the same neuron shown in A before addition of N/OFQ and after a steady-state effect had been reached. Note that N/OFQ did not affect the amplitude of mIPSCs, but shifted mIPSC inter-event intervals to larger values. D, time course of the N/OFQ effect. E and F, relative mIPSC amplitude (E) and frequency (F) pooled during control conditions and after addition of N/OFQ demonstrate a significant decrease in mIPSC frequency to 84.4 %, whereas the mean amplitude was left unchanged.
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DISCUSSION |
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Involvement of ORL receptors
The present results demonstrate an inhibitory action of N/OFQ at concentrations in the nanomolar range on excitatory and inhibitory synaptic transmission in the majority of putative projection neurons in the rat lateral amygdala. In general, effects of N/OFQ were not reversible, which may be attributable to slow dissociation of the ligand (Ardati et al. 1997) and/or slow washout of the drug during bath perfusion (Muller et al. 1988). The action of N/OFQ seems to be specific to the ORL receptor, since the effect was concentration dependent and was reduced by PheN/OFQ. The concentration range at which N/OFQ modulated EPSC and IPSC amplitudes is similar to the N/OFQ-mediated increase in postsynaptic K+ conductance previously described in this preparation (Meis & Pape, 1998), and is comparable to the action of N/OFQ on release and synaptic transmission in various regions of the brain (Calo' et al. 2000). PheN/OFQ has since been reported to act as antagonist, partial agonist or even full agonist of ORL receptors (Calo' et al. 2000). These controversial results were postulated to depend on differences between central versus peripheral functional sites (Okawa et al. 1999), human versus rodent ORL receptors (Emmerson & Miller, 1999), variations in receptor densities (Toll et al. 1998) or stimulus-response efficiencies (Calo' et al. 2000). In rat LA neurons, application of PheN/OFQ at concentrations 5 µM induced a K+ outward current (S. Meis, unpublished observations), in line with reports describing PheN/OFQ as a partial agonist of the ORL receptor. In the present study, no agonistic effects on EPSC amplitudes were detected by 1 µM PheN/OFQ. On the other hand, modulatory effects by N/OFQ were reduced during the presence of 1 µM PheN/OFQ, confirming the involvement of ORL receptors.
Site and mechanisms of N/OFQ action
While N/OFQ has been reported to inhibit neurotransmitter release in various preparations, due to a reduction in excitability of the presynaptic neuronal elements and/or a reduction in presynaptic transmitter secretion (Calo' et al. 2000), only limited information is available on the influences exerted by N/OFQ on evoked postsynaptic currents. For instance, N/OFQ (300 nM) reduced evoked glutamatergic EPSCs and GABAergic IPSCs by 46 and 49 %, respectively, in rat periaqueductal grey neurons (Vaughan et al. 1997). Evoked EPSCs in superficial dorsal horn neurons of the rat spinal cord were suppressed by roughly 50 % with N/OFQ with a half-maximal effective concentration of 485 nM, while IPSCs were unaffected (Liebel et al. 1997; Zeilhofer et al. 2000). Finally, 100 nM to 1 µM N/OFQ led to a reduction in EPSC amplitudes by around 50 % in arcuate nucleus neurons, while it exerted no effect in the ventromedial hypothalamus (Emmerson & Miller, 1999). Similar to those preparations, the effect of N/OFQ was only partial in the LA, presumably reflecting a proportion of stimulated terminals unresponsive to N/OFQ and/or a mechanism of action that is only partially effective in suppressing transmitter release.
Several lines of evidence indicate that N/OFQ exerts its suppressant action on synaptic transmission in the LA through a presynaptic mechanism, thereby voting against a contamination by postsynaptic effects of N/OFQ. All experiments were performed with GDP--S added to the intracellular solution, resulting in a reliable block of N/OFQ-mediated activation of G-protein-coupled K+ channels (Meis & Pape, 1998), as confirmed through monitoring of the holding current during the course of the experiments. One possible point of concern relates to distally located K+ channels, which, due to limited diffusion of GDP--S into dendritic compartments, might not have been reached. This concern seems particularly valid in view of distal glutamatergic inputs known to exist in amygdaloid neurons (Farb & LeDoux, 1999). The following evidence argues against this possibility. If distally located K+ channels were not reached, activation of these channels through N/OFQ would have led to a change in holding current. This was not observed. If non-blocked dendritic K+ channels were electrotonically uncoupled from the somatic recording site (resulting in a lack of change in somatic holding current despite activation of those channels), modulation of those channels and the resulting effects on nearby synaptic signals would have also escaped somatic recording, thereby not contaminating analyses of more proximal synaptic events. Furthermore, N/OFQ reduced the frequency but did not affect the amplitude of spontaneous, TTX-independent miniature postsynaptic currents, which also votes in favour of an action on receptors located at or near the presynaptic terminals, and against a modulation of postsynaptic functions. In summary, it seems feasible to conclude that the effects of N/OFQ on IPSCs and EPSCs reported in the present study reflect a presynaptic site of action.
Mechanisms suggested to be involved in the regulation of transmitter release through presynaptic receptors include inhibition of Ca2+ channels within the nerve terminal, activation of presynaptic K+ channels or direct modulation of the release apparatus (Miller, 1998). mEPSCs are usually thought to result from the spontaneous exocytosis of transmitter-containing vesicles occurring in the absence of Ca2+ influx (Miller, 1998). Indeed, the application of Cd2+ at a concentration (200 µM) known to block Ca2+ influx via voltage-dependent Ca2+ channels in amygdaloid neurons (Viana & Hille, 1996; Yu & Shinick-Gallagher, 1998), did not affect mEPSC amplitude or frequency. Therefore, the observation of quantitatively equivalent inhibition of mEPSCs (74.0 %) and evoked EPSCs (70.3 %), displaying a similar magnitude and time course, and occurring in a comparable fraction of neurons, may argue in favour of mechanisms operating downstream of Ca2+ entry (Wu & Saggau, 1997). By comparison, the N/OFQ-induced inhibition of evoked and spontaneous GABAergic responses by N/OFQ was not quantitatively equivalent. Besides effects exerted directly on or downstream of Ca2+ entry into the nerve terminal, a hyperpolarization through activation of ORL-coupled K+ channels located in the somatodendritic membrane of interneurons is conceivable. This would at least partly prevent their excitation by the focal stimulus, leading to reduced spike activity and associated reduction of GABA release.
Lack of effect of nocistatin in LA neurons
Another product of the N/OFQ precursor peptide prepronociceptin was named nocistatin, taking into account its activity as a functional antagonist of N/OFQ (Okuda-Ashitaka et al. 1998). Nocistatin indeed counteracted N/OFQ effects in pain transmission in vivo (Okuda-Ashitaka et al. 1998; Zhao et al. 1999; Xu et al. 1999; Nakano et al. 2000), and in learning and memory (Hiramatsu & Inoue, 1999). In vitro, nocistatin reversed the inhibition of glutamate release by N/OFQ from rat brain slices (Nicol et al. 1998). The cellular basis of these effects is not yet established. A recent study described a suppression of inhibitory synaptic transmission mediated through GABA and glycine receptors by nocistatin in the rat spinal dorsal horn, acting via a presynaptic mechanism, whereas N/OFQ affected excitatory synaptic transmission only (Zeilhofer et al. 2000). The fine-tuned modulation of synaptic mechanisms by N/OFQ in concert with nocistatin seems not to be mirrored in the amygdala. Here, nocistatin had no measurable effect on intrinsic membrane or synaptic properties in putative projection cells whereas N/OFQ affected excitatory as well as inhibitory synaptic transmission (present study) and stimulated a G-protein K+ conductance (Meis & Pape, 1998). Similar to these findings, nocistatin did not affect the membrane properties of rat locus coeruleus neurons (Connor et al. 1999) or rat hippocampal CA3 neurons (Amano et al. 2000), and did not display any antagonist action regarding the inhibition of calcium currents (Connor et al. 1999) or the activation of outward K+ currents (Amano et al. 2000) by N/OFQ.
Functional considerations
The expression of ORL mRNA and the receptor protein, as determined through in situ hybridization, autoradiographic and immunocytochemical techniques, is very dense to dense in the amygdala (Darland et al. 1998). The N/OFQ peptide is differentially distributed among the various amygdaloid subnuclei, ranging from intense in the central and medial amygdala to undetectable in the LA (Neal et al. 1999b). These results seem to be at odds with our present and previous (Meis & Pape, 1998) findings that the majority of LA neurons are responsive to N/OFQ through pre- and postsynaptic mechanisms. However, functional ORL1 receptors have been reported to exist in the LA (Neal et al. 1999a), although their exact cellular location and the patho/physiological conditions under which N/OFQ is produced or released remain to be evaluated. The reason for the lack of N/OFQ detection in the LA is not known, but may relate to a low overall concentration of the peptide, a diffuse distribution of the peptide relating to non-synaptic interactions, a differential regulation of peptide synthesis, or a combination thereof.
Functionally, the amygdala plays a central role in the emotional interpretation of sensory information, with the LA representing the major sensory input station (Pitkänen et al. 1997). Glutamate is considered to be the excitatory transmitter of sensory information that is relayed to the LA, for instance, through cortical, thalamic and hippocampal input fibres (LeDoux, 2000). In addition, glutamatergic transmission to projection neurons in lateral and basolateral amygdaloid nuclei has been found to be regulated on a long-term scale through activity-dependent synaptic plasticity, which recruits both pre- and postsynaptic mechanisms (Fendt & Fanselow, 1999; Maren, 1999). In view of the proposed role of synaptic plasticity within the amygdala for conditioned fear (LeDoux, 2000) and the reported anxiolytic effects of N/OFQ during fear-related behaviour (Jenck et al. 1997; Griebel at al. 1999), it is interesting to speculate that the inhibitory regulation of glutamate release to LA projection neurons through N/OFQ may represent a mediating cellular mechanism. A second point of interest relates to the N/OFQ-mediated control of GABAergic transmission, particularly in light of the reported dominance of inhibitory influence on neuronal responsiveness in the amygdala (Lang & Paré, 1997). Overall, the amygdala seems to be equipped with an inhibitory gating mechanism regulating information flow from afferent cortical and thalamic input fibres via projection neurons (Szinyei et al. 2000) through intra-amygdaloid circuits (Lang & Paré, 1998). N/OFQ, in turn, may participate in these neuronal processes by interacting with the GABAergic interneurons or directly with the GABA release machinery, thereby controlling the inhibitory mechanisms and/or modulating the potentiation of GABAA-mediated synaptic currents (Mahanty & Sah, 1998).
Finally, it is interesting to note that N/OFQ and classical opioids seem to share a number of common signalling pathways, although specific differences and even opposing effects are exerted on the systemic level via ORL and opioid receptors in a number of functional subsystems (Harrison & Grandy, 2000). In the LA, an opioid receptor-mediated hyperpolarization and a presynaptic regulation of IPSCs, but not EPSCs, occurred predominantly in non-pyramidal types of cells (Sugita & North, 1993; Sugita et al. 1993), while ORL activation was associated with an increase in postsynaptic K+ conductance (Meis & Pape, 1998) and a reduction of both EPSCs and IPSCs (present study). Therefore it appears as if different types of neuronal pathways in the LA are associated with classical opioid receptor and ORL receptor-mediated effects. Future studies are needed to evaluate the significance of this cellular situation for pain- and fear-related behaviour (Yamamoto et al. 1999).
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Acknowledgements
We thank Dr Szinyei for participating during some initial experiments. Thanks are due to R. Ziegler for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 426, TP B3) and Kultusministerium des Landes Sachsen-Anhalt (2278A/0085).
Corresponding author
H.-C. Pape: Institut für Physiologie, Medizinische Fakultät, Otto-von-Guericke-Universität, Leipziger Strasse 44, D-39120 Magdeburg, Germany.
Email: hans-christian.pape{at}medizin.uni-magdeburg.de
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