Nociceptin reduces epileptiform events in CA3 hippocampus via presynaptic and postsynaptic mechanisms

Abstract

The opiate-like peptide nociceptin/orphanin FQ (Noc) and its receptor [opiate receptor-like receptor (ORL-1)] are highly expressed in the hippocampus. Noc has inhibitory postsynaptic actions in CA1, CA3, and the dentate and seems to lack the disinhibitory, excitatory actions demonstrated for some opiate peptides in the hippocampus. The CA3 hippocampal region is important in the generation of hippocampal seizures. Therefore, we tested the action of Noc on spontaneous epileptiform activity recorded extracellularly or intracellularly in CA3 and generated by removal of Mg(2+) from the bathing solution or by raising extracellular K(+) from 3.5 to 7.5 mm. Superfusion of Noc robustly depressed spontaneous bursting without desensitization. The ORL-1 antagonist [Phe(1)Psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2) (1-2 microm) greatly attenuated the reduction of spontaneous bursting by Noc. To characterize the cellular mechanism of action of Noc, we recorded intracellularly from CA3 pyramidal neurons. Noc reduced EPSCs evoked by stimulating either mossy or associational/commissural fibers. Analysis of miniature EPSCs using whole-cell voltage-clamp recording suggests that Noc acts presynaptically to inhibit glutamate release. This is the first demonstration of a presynaptic effect for Noc in the hippocampus. Noc also increased K(+) currents in CA3 pyramidal neurons, including the voltage-sensitive M-current. Blocking the M-current with linopirdine increased the duration of individual CA3 bursts but did not attenuate Noc-mediated inhibition of bursting. Thus, Noc acts via multiple mechanisms to reduce excitation in CA3. However, Noc inhibition of epileptiform events is not dependent on augmentation of the M-current.

Fig. 1.

Noc reduced spontaneous bursting recorded in CA3. Extracellular recordings in Mg²⁺-free ACSF. In this slice, Noc reduced the burst rate from 0.3 to 0.07 Hz, with recovery after washout (Wash). This effect was blocked by coapplication of ORLAn, the ORL-1 antagonist. Inhibition of bursting during a subsequent application of Noc (1–13) amide showed that no desensitization occurred. Bottom right, An individual burst with an expanded time base. Noc did not consistently affect the shape of individual bursts (data not shown).

Fig. 2.

Noc reduced MF-generated EPSCs.A–C, Plots of mean EPSC amplitudes versus stimulation strength are shown. A, Noc (500 nm;closed squares) reduced the mean amplitude of EPSCs generated by stimulating MFs in the stratum radiatum. This effect reversed after washout (open circles;n = 6–9 cells). B, A lower concentration of Noc (200 nm) also reduced the mean amplitude of MF EPSCs (closed squares;n = 5). This effect was blocked when Noc was coapplied with 1 μm ORLAn (open triangles;n = 5). C, The truncated analog Noc (1–13) amide (500 nm; closed squares) also effectively reduced mean MF EPSC amplitude (n = 5), with complete recovery after washout (open circles).D, Representative current traces recorded from two CA3 neurons are shown. Top row, MF EPSCs generated at half-maximal stimulus intensity were attenuated by 200 nmNoc, with recovery after washout. Bottom row, In a different neuron, ORLAn (1 μm) alone did not alter the evoked EPSC but blocked the effect of 200 nm Noc when coapplied. Noc and Noc with ORLAn were tested in different neurons to avoid desensitization issues. Con, Control;Max, maximal; OrlAn, ORLAn;Thresh, threshold.

Fig. 3.

Noc reduced A/C (AC)-generated EPSCs. A–C, Mean EPSC amplitudes versus stimulation strength are shown. A, Noc (500 nm;closed squares) reversibly reduced the mean amplitude of EPSCs generated by stimulating A/C fibers in the stratum radiatum. This effect was reversible after washout (open circles;n = 7–10). B, A lower concentration of Noc (200 nm) also reduced the amplitude of A/C EPSCs (closed squares; n = 5). This effect was completely blocked when Noc was coapplied with 1 μmORLAn (open triangles; n = 5).C, Noc (1–13) amide (500 nm) also effectively reduced mean EPSC amplitude (closed squares;n = 5), with recovery after washout (open circles). D, Representative current traces from CA3 neurons show A/C EPSCs. Top row, Noc (200 nm) reduced the EPSC generated at half-maximal stimulus intensity, an effect that washed out. Bottom row, In contrast, in a different neuron, 1 μm ORLAn alone or coapplied with 200 nm Noc did not alter the evoked A/C EPSCs.

Fig. 4.

Noc reduced the frequency of mEPSCs.A, Whole-cell voltage-clamp recordings from a representative CA3 neuron in TTX and bicuculline. Note the Noc-induced decrease in the frequency of the mEPSCs. B, Cumulative frequency histogram for a representative neuron showing a shift to longer interevent intervals (lower frequencies) after application of Noc (500 nm). Data were plotted in 25 msec bins and averaged from three different 20 sec recording intervals each for control and Noc. C, Cumulative amplitude graph from the same neuron showing no change in the distribution of mEPSC amplitudes. Data shown are means from three 20 sec recordings plotted in 1 pA bins.D, Pooled data showing mean inhibition of mEPSC frequency by 500 nm Noc (n = 5). Theasterisk denotes statistical significance (p < 0.05). E, Mean amplitudes of mEPSC from the same five neurons. Noc (500 nm) did not significantly affect mean mEPSC amplitude.Prob, Probability.

Fig. 5.

Intracellular voltage-clamp recordings from CA3 pyramidal neurons showing current–voltage relationships of Noc effects. A, Current traces from a representative neuron. Superfusion of 0.5 μm Noc (5 min) increased steady-state currents across the range of voltages tested, with recovery after washout (21 min). RMP was −70 mV, and V_Hwas −62 mV. Voltage commands are shown at lower left.B, left, Current–voltage (I–V) plot for mean net (control-subtracted) current elicited by 0.5 μm Noc (n = 5). The nociceptin-induced current reversed at −97 mV, suggesting that the Noc current is carried by K⁺ ions. Right, Mean control-subtracted currents for ORLAn alone (1 μm) and for 0.5 μm Noc plus 1 μm ORLAn (n = 6–7). Note that at this concentration, ORLAn has very little partial agonist activity but blocks almost all of the action of Noc.

Fig. 6.

Noc increased the M-current in CA3 neurons; this effect was blocked by linopirdine. A, M-current current record of a CA3 neuron in the presence of 10 μm nifedipine and 1 μm TTX is shown. Superfusion of 0.5 μm Noc for 6 min induced an outward current and increased the M-current with recovery after washout (34 min). Voltage command steps are shown at lower left;V_H = −49 mV; RMP = −71 mV.B, I–V analysis of pooled data from five cells shows a significant Noc enhancement of M-current amplitudes with complete recovery after washout. C, In another cell 10 μm linopirdine (Lino), an M-current blocker, induced a small inward baseline current consistent with the observed blockade of the M-current (note flat current traces). When superfused with linopirdine, 0.5 μm Noc had no effect on the M-current but still induced a small steady-state outward current.Inset, The middle current for linopirdine subtracted from the control current isolates the linopirdine-sensitive component (M-current). Scale units are the same as for A. Voltage command steps are shown at lower left;V_H = −40 mV; RMP = −67 mV.D, Analysis of pooled, control-subtracted steady-state values from five cells indicates that Noc still induced a significant, but greatly reduced, outward current in linopirdine. The Noc-induced steady-state current (without linopirdine) is from the same five cells shown in B. The residual Noc-induced current in linopirdine reversed near the K⁺ equilibrium potential. These findings suggest that Noc activates two different K⁺ currents.

Fig. 7.

Linopirdine did not attenuate Noc inhibition of burst frequency in epileptiform models. A, Representative voltage traces recorded extracellularly in the CA3 cell layer are shown. In Mg²⁺-free ACSF containing linopirdine (10 μm; 30 min), Noc (0.5 μm) was still able to block spontaneous bursting completely, with recovery after washout. After Noc superfusion, membrane potential was manually adjusted to the control level with current injection.Inset, Spontaneous extracellular bursts in Mg²⁺-free ACSF are shown with an expanded time scale. Calibration: 100 msec, 0.5 mV. Application of linopirdine (30 min) resulted in an increase in the duration of the burst, with the appearance of multiple secondary afterdischarges. B, Extracellular recordings show that Noc (0.5 μm) still had a full inhibitory effect in linopirdine when high extracellular K⁺ (7.5 mm) was used to induce spontaneous bursting. C, Intracellular current-clamp recordings of spontaneous bursting in 0.75 mmMg²⁺ and picrotoxin (50 μm) are shown.Top, In the presence of linopirdine, Noc completely blocked bursting at the RMP (−74 mV; current injection was used to keep the membrane potential constant after Noc application).Bottom, Even when the neuron was depolarized by 14 mV with current injection, blocking M-currents with linopirdine did not interfere with the ability of Noc to suppress bursting. Note that bursts can be identified by large afterhyperpolarizations. When the neuron was depolarized beyond the threshold for action potentials, single spikes are observed that Noc did not affect (membrane potential was held constant after Noc application by current injection).Inset, Single spontaneous burst recorded intracellularly is shown with an expanded time scale. Calibration: 250 msec, 25 mV. As with extracellular recordings, linopirdine (35 min) increased the duration of individual bursts and increased the number of afterdischarges.