Ž .Brain Research 853 2000 269–274www.elsevier.comrlocaterbres

Research report

Orphanin FQ-induced outward current in rat hippocampus

Taku Amano a, Hiroaki Matsubayashi a, Yutaka Tamura b, Toku Takahashi c,)

a Department of Pharmacology, Hiroshima UniÕersity School of Medicine, Hiroshima, Japanb Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama UniÕersity, Fukuyama, Japan

c Department of Internal Medicine, 6520 Medical Science Research Building I, The UniÕersity of Michigan, 1150 W. Medical Center DriÕe, Ann Arbor,MI 48109-0682, USA

Accepted 19 October 1999

Abstract

Ž .Orphanin FQ OFQ is a heptadecapeptide that structurally resembles opioid peptides. It has been demonstrated that the moderatedensity of binding sites of OFQ were localized in the hippocampus and that the expression of OFQ receptor in the hippocampus have animportant role in learning and memory. This study was designed to investigate whether activation of the OFQ receptor could induced

Ž y8 y5 .hyperpolarization in the cultured hippocampus neurons in rats. In the current clamp mode, the application of OFQ 10 –10 Mhyperpolarized the membranes in cultured hippocampus neurons in a concentration-dependent manner. Moreover, in the voltage clamp

Ž y6 . Ž y7mode, application of OFQ 10 M induced outward current in hippocampus CA3 pyramidal neurons. In the presence of TTX 3=10. Ž y6 .M , the average maximal amplitude of the outward current deflection induced by OFQ 10 M at y60 mV of a holding potential was

Ž . q24.7"0.54 pA. The OFQ-induced current reversed at y99.06"3.80 mV 3 mM , which was quite close to the K equilibrium potentialŽ . qas calculated by the Nernst equation E sy96.08 mV, 3 mM for K in our standard solution. This suggests that OFQ-induced currentk

q w 1 Ž . 2 x Ž . . Žwas mediated by K ion. It has been demonstrated that Phe c CH -NH Gly Nociceptin 1–13 NH a pseudopeptide analog of2 2. Ž y6 .nociceptin , and nocistatin are selective antagonists of OFQ. OFQ 10 M -induced outward current was antagonized by application of

w 1 Ž . 2 x Ž . Ž y5 .Phe c CH -NH Gly Nociceptin 1–13 NH 10 M . In contrast, OFQ-induced outward current was not antagonized by application2 2Ž y5 .of nocistatin 10 M . These results indicates that there is the physiological functioning receptor of OFQ in the hippocampus. q 2000

Elsevier Science B.V. All rights reserved.

w 1 Ž . 2 x Ž .Keywords: Hyperpolarization; Patch clamp; Nocistatin; Phe c CH -NH Gly Nociceptin 1-13 NH2 2

1. Introduction

During several years, several groups have isolated andcloned a new G protein-coupled receptor that showed highhomology with opioid receptors. None of the known opi-oid ligands was found to bind to this receptor, which wastherefore considered as an orphan member of the opioid

w xreceptor family. Two independent groups 11,14 identifieda 17 amino acid long peptide that did not bind to theclassical opioid receptors but activated the orphan receptor.This peptide was named as orphanin FQ by Reinscheid et

w x w xal. 14 and nociceptin by Meunier et al. 11 and Mollereauw xet al. 12 .

OFQ acts in the brain to produce various pharmacologi-cal effects, such as hyperalgesia and hypolocomotion. Ithas been demonstrated that the high or moderate density of

) Corresponding author. Fax: q1-734-763-2535; e-mail:ttakahas@umich.edu

w3 xbinding sites of H OFQ were localized in the centralnervous system including the hippocampus of the mousew x5 . Histological analysis revealed the expression of bothOFQ precursor and OFQ receptor in the hippocampus havean important role in learning ability. The microinjection ofOFQ into the CA3 region of the dorsal hippocampusseverely impaired spatial learning and markedly decreased

w xexploratory locomotor activity 16 . It has recently beendemonstrated that mice lacking OFQ receptor possessgreater learning ability and have better memory than con-

w xtrol mice 10 .Although the OFQ-immunopositive neurons and its re-

w xceptors are abundant in the hippocampus 5,10 , in vitropharmacological evaluation of OFQ still remains unknown.The present study was designed to investigate whetheractivation of OFQ receptor could induced hyperpolariza-tion in the hippocampus.

It has been shown that nocistatin, a new biologicallyactive peptide produced from the same precursor as OFQ,

0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 02245-3

( )T. Amano et al.rBrain Research 853 2000 269–274270

w xblocks OFQ-induced allodynia and hyperalgesia 13 . Aw 1 Ž . 2 xpseudopeptide analog of OFQ, Phe c CH -NH Gly2

Ž . 2Nociceptin 1–13 NH , has also been proposed as a selec-tive antagonist of OFQ in the guinea pig ileum and mouse

w xvas deferens 7 . However, it still remains unknown whetherthese putative OFQ antagonists have inhibitory effects onOFQ-induced responses in the hippocampus.

2. Materials and methods

2.1. Cell culture

Primary cultures were prepared from fetal rats on the16–18 day gestation. The anterior hippocampus was re-moved bilaterally according to the method described by

w xFreese et al. 6 , with minor modifications. The hippocam-pus was minced, mechanically dissociated using scalpel

Ž .blades, filtered using a stainless steel mesh 150 mesh ,and plated as single-cell suspensions on plastic or glass

Žcoverslips that were placed in Falcon 60 mm dishes ap-6 .proximately 4.5=10 cellsrdish . In each dissecting ses-

Ž .sion, 3–4 litters 10–15 pupsrlitter were used. Thew xcultures were maintained as described previously 1,18 .

Cultures were incubated in Eagle’s minimal essentialŽ .salt medium Eagle’s MEM supplemented with 10%

Žheat-inactivated fetal bovine serum 1–9 days after plat-. Žing or 10% heat-inactivated horse-serum 10–12 days

. Ž . Ž .after plating , glutamate 2 mM , glucose total 11 mM ,Ž . Ž .NaHCO 24 mM , and HEPES 10 mM . Cultures were3

maintained at 378C in a humidified 5% CO atmosphere.2

After 8 days of plating, non-neuronal cells were removedby adding 10 mM cytosine arabinoside. Cultures main-tained 10–14 days in vitro were used for degeneration andpatch clamp studies.

2.2. Whole cell experiment of cultured neurons

Whole cell currents were recorded according to thew xstandard patch clamp techniques 4,8,15,17 . Under the

current-clamp mode, membrane potentials were recordedfrom these hippocampus CA3 neurons according to thestandard whole-cell patch-clamp technique using an Ax-

Žopatch 200A patch-clamp system Axon Instruments, Fos-.ter City, CA . The ionic composition of the artificial

cerebro-spinal fluid for dissection and perfusion was asŽ .follows in mM : NaCl, 113; KCl, 3; NaH PO , 1;2 4

Ž .NaHCO , 25; glucose, 11; CaCl , 2; MgCl , 1 pH 7.4 .3 2 2

The pH of the solution was 7.4 when bubbled with O –CO2 2Ž .95–5% . The recording chamber was perfused at 4mlrmin. The drugs were applied into the bath by switch-ing the perfusion line manually. The dead space time wasabout 20 s. Experiments were carried out at room tempera-ture. The patch microelectrode had resistance of 5–8 MV.No series resistance compensation was used in this study.Patch pipette was made from standard-walled borosilicate

Ž .glass capillaries Clark, o.d. 1.5 mm , their tips beingcoated with sylgard to reduce stray capacitance across theglass wall and fire-polished. The liquid-junction potentialbetween the pipette and standard Krebs solution was cor-rected at the beginning of the experiment.

2.3. Slice patch-clamp study

Ž .After decapitation of young rats 12–17 days old , ablock of tissue containing the corpus hippocampus wastrimmed and kept in ice-cold artificial cerebro-spinal fluid

Ž . Žbubbled with O –CO 95–5% . The slices 150 mm2 2.thickness including corpus striatum were cut coronally

Ž .from the block using a microslicer Dosaka, DTK-1000and were incubated at 348C for 1 h before experiments. Onthe experiments, the hippocampus slices were placed in arecording chamber perfused continuously with artificialcerebro-spinal fluid at 2 mlrmin. The ionic composition ofthe artificial cerebro-spinal fluid for dissection and perfu-

Ž .sion was as follows in mM : NaCl, 113; KCl, 3; NaH PO ,2 4

1; NaHCO , 25; glucose, 11; CaCl , 2; MgCl , 1;3 2 2Ž .tetrodotoxin TTX 0.0003. The pH of the solution was 7.4

Ž .when bubbled with O –CO 95–5% . OFQ, nocistatin2 2w 1 Ž . 2 x Ž .and Phe c CH -NH Gly Nociceptin 1-13 NH were2 2

dissolved from a 10 mM stock solution frozen in aliquots,and was applied into bath by switching the perfusion linemanually. The hippocampus CA3 pyramidal cells wereidentified under high magnification of the Nomarski optics

Ž .with a water-immersion lens =600 Olympus BX50WI .Under the voltage-clamp mode, membrane currents were

recorded from these hippocampus CA3 neurons accordingto the standard whole-cell patch-clamp technique using an

Ž .EPC 8 patch-clamp system HEKA Elektronik: Germany .Patch pipettes were pulled from borosilicate capillary glassŽ .Clark, o.d. 1.5 mm and had resistances between 5–8 MV

when filled with internal solution which had the followingŽ .ionic composition in mM : K-gluconate, 130; KCl, 10;

ŽCaCl , 1; HEPES, 10; EGTA, 10 mM pH adjusted to 7.32.with 4 mM KOH . The signals of membrane current

filtered at 3 kHz by bessel filter were monitored on theŽ .oscilloscope Nihon Kohden, VC-11 and recorded directly

Ž .on Thermal Array Recorder Nihon Kohden, RTA-1100and sampled by a microcomputer using the program

Ž .pClamp7 Axon Instruments, Foster City, CA or storedfor off-line analysis on videocassette tapes after passage

Žthrough a pulse code modulation device Instutech, VR-.10B, Great Neck, NY . To obtain the I–V relationship of

the current induced by OFQ, ramp-shaped voltage com-Ž .mands from y120 to y30 mV; 20 mVrs were applied

before and during drug administration.

2.4. Chemicals

The following drugs and sources were used. OFQ,Ž . w 1 Žnocistatin Phoenix, Mountain View, CA , Phe c CH -2

. 2 x Ž . Ž .NH Gly Nociceptin 1-13 NH Tocris, Ballwin, MO and2

( )T. Amano et al.rBrain Research 853 2000 269–274 271

Ž .tetrodotoxin TTX . The drugs were dissolved in artificialcerebro-spinal solution immediately before the experi-ments.

2.5. Statistical analysis

All data were expressed as means"S.E. The peakamplitude of the whole-cell currents and voltage weredetermined using the pCLAMP7 program. Statistical anal-ysis was performed by paired t-test.

3. Results

3.1. Whole cell experiment of cultured hippocampus neu-rons and slice preparation under the current-clamp mode

Whole-cell recordings were made from 18 visuallyidentified cultured hippocampus neurons, bath applicationof OFQ induced hyperpolarization in 13 of 18 neuronsŽ .72.2% , the diameter of which was more than 17.8 mmŽ .ns13 . The mean resting membrane potential of neuronsrecorded in the present study were y50.6"3.06 mVŽ .ns13 . Under current-clamp conditions, bath applicationof OFQ hyperpolarized the membrane potential of cultured

Ž .hippocampus neurons Fig. 1B . The mean amplitude ofŽ y7 .hyperpolarization induced by OFQ 10 M was in-

creased in dose-dependent manner. The mean amplitude ofhyperpolarization induced by 10y8 , 10y7, 10y6 and 10y5

Ž .M OFQ was y2.4"0.67 mV ns4 , y7.4"0.89 mV

Ž .Fig. 1. A Concentration–response curves for the hyperpolarizationŽ y8 y5 .induced by OFQ 10 –10 M on the cultured hippocampus neurons

Ž .A . Each circle and error bar represents the mean with and S.E.. Numberof neurons tested in parentheses. EC values for OFQ and Hill slopes50

Ž .were 0.17"0.20 mM and 0.70"0.04, respectively. B HyperpolarizingŽ y6 . y7effect of bath application of OFQ 10 M at a concentration of 10

M on the membrane potential and spontaneously occurring action poten-tials of hippocampal neuron. The period during drug application isindicated by horizontal bar.

Fig. 2. OFQ-induced Kq currents in hippocampal CA3 pyramidal cells.The membrane current was recorded from CA3 pyramidal cell using

Ž y6 .whole-cell voltage clamp mode. OFQ 10 M caused an outwardŽ .current at y60 mV of the holding potential A . I –V curve 1 was

Ž .subtracted from I –V curve 2 B to obtain the I – V relationship of theŽ .OFQ-induced net current C .

Ž . Ž .ns8 , y14.5"1.09 mV ns4 and y17.4"1.15 mVŽ . Ž .ns3 , respectively Fig. 1A . The data points were bestfitted by a sigmoidal function. The estimated EC value50

and Hill slope were 0.17"0.20 mM and 0.70"0.04,respectively. Same hyperpolarization was observed in theslice experiment in current clamp mode. This hyperpolariz-ing effects of OFQ on hippocampal neurons were notdifferent between the slice preparations and cultured neu-rons.

3.2. OFQ-induced actiÕation of K q channel in hippocam-pus CA3 neurons

Recordings were derived from a total of 43 visuallyidentified hippocampal CA3 pyramidal neurons in the slicepreparations. The mean of long and short axis diameter

Ž .was 16.34"0.30 ns43 . The mean resting membranepotential of neurons recorded in the present study were

Ž .y52.8"1.32 mV ns43 . Bath application of OFQŽ y6 .10 M induced outward current in 39 of 43 neuronsŽ .90.7% , although there was no effect of OFQ other 4

Ž y7 .neurons. In the presence of TTX 3=10 M , the aver-age maximal amplitude of the outward current deflection

Ž y6 .induced by OFQ 10 M at y60 mV of holding poten-Ž . Ž .tial was 24.7"0.54 pA Fig. 2A ns28 . Similar re-

sponses to OFQ were observed during blockade of synap-tic transmission in the presence of TTX and Ca2q-free

Ž .solution 23.83"0.96 pA; ns4 .

( )T. Amano et al.rBrain Research 853 2000 269–274272

ŽRamp-shaped voltage commands from y120 to y30.mV; 20 mVrs were applied to monitor the I–V relation-

Žship of the recorded cell in the control period Fig. 2B,. Žcurve 1 and during the application of OFQ Fig. 2B, curve

.2 . The I–V relationship of the net current induced byOFQ was obtained by subtracting curve 2 from curve 1Ž .Fig. 2C, curve 2-1 .

OFQ-induced net current reversed at y99.06"3.80Ž . qmV 3 mM, ns4 which were quite close to the K

equilibrium potential as calculated by the Nernst equationŽ . qE sy96.08 mV, 3 mM for K in our standard solu-k

tion. When Kq concentration of the perfusing solutionincreased at 6 mM and 9mM, the reversal potential, also

Ž .were increased at y78.2"1.28 6 mM, ns3 andŽ .y68.1"1.67 9 mM, ns4 , respectively. The shift of

reversal potential was followed by Nernst equation, al-Ž .though the regression line solid line deviated slightly

Ž . Ž .from the predicted position dotted line Fig. 3 . This maybe due to voltage errors caused by the series resistance.

1 ( ) 2] ( )3.3. Effects of Phe c CH -NH gly Nociceptin 1–13 NH2 2

and nocistatin on OFQ-induced outward current

It has been shown that nocistatin, a new biologicallyactive peptide produced from the same precursor as OFQ,

w xblocks OFQ-induced allodynia and hyperalgesia 13 . Aw 1 Ž . 2 xpseudopeptide analog of OFQ, Phe c CH -NH Gly2

Ž .Nociceptin 1–13 NH , has also been shown as a selective2

antagonist of OFQ receptor in the guinea pig ileum andw xmouse vas deferens 7 .

1 ŽTo investigate whether nocistatin and Phe c CH -2. 2 x Ž .NH Gly Nociceptin 1–13 NH have antagonistic effects2

on OFQ-induced outward current in hippocampus CA31 Žneurons, we studied effects of nocistatin and Phe c CH -2

. 2 x Ž .NH Gly Nociceptin 1–13 NH on OFQ-induced outward2

Fig. 3. The reversal potential at various external Kq concentrations inhippocampal CA3 pyramidal cells. The reversal potential was measuredat 3, 6 and 9 mM of external Kq. In each slice, only one Kq concentra-tion was examined. When Kq concentrations was increased, a corre-sponding amount of NaCl was replaced with KCl. The solid line indicatesa linear regression line fitted with the least-squares method. The dotted

Žline indicates the relationship predicted by Nernst equation mean"S.E.,.ns3–4 .

w 1 Ž . 2 x Ž .Fig. 4. Effects of Phe c CH -NH Gly Nociceptin 1–13 NH and2 2

nocistatin on OFQ-induced outward current. The membrane current wasŽ .recorded at y60 mV. OFQ-induced outward current A was antagonized

w 1 Ž . 2 x Žby concomitant application of Phe c CH -NH Gly Nociceptin 1–2. Ž .13 NH B . In contrast, OFQ-induced outward current was not affected2

Ž .by concomitant application of nocistatin C .

current. It has been generally accepted that voltage clampmode is much more sensitive than current clamp mode to

w xmeasure ion-channel activity 9,15 . Both of nocistatinŽ y 5 . 1 Ž . 2 x Ž10 M and Phe c CH -NH Gly Nociceptin 1–2. Ž y5 .13 NH 10 M themselves had noeffects on holding2

currents at y60 mV. This suggests that nocistatin andw 1 Ž . 2 x Ž .Phe c CH -NH Gly Nociceptin 1–13 NH have no ef-2 2

fects on membrane potential in current clamp mode. OFQŽ y6 . Ž10 M -induced outward current 23.43"1.02 pA, ns. Ž7 was significantly reduced to 2.86"0.86 pA ns7,

. 1 Ž . 2 xP-0.01 in the presence of Phe c CH -NH Gly Noci-2Ž . Ž y5 . Ž .ceptin 1–13 NH 10 M Fig. 4B . On the other hand,2

y6 Žthe outward current induced by OFQ 10 M 23.93"0.80.pA, ns14 was not affected in the presence of nocistatin

y6 Ž . y5 Žat 10 M 23.50"1.36 pA, ns6 , 10 M 23.25". y4 Ž1.97 pA, ns4; Fig. 4C and 10 M 22.751"2.32 pA,

.ns4 .

4. Discussion

In our present study, bath application of OFQ-induceddose-dependent hyperpolarized the membrane of culturedhippocampus neurons. This hyperpolarization was re-cognized with hippocampal slice preparations. In vol-tage-clamp conditions, bath application of OFQ inducedoutward current in almost 90% of visually identifiedhippocampus CA3 pyramidal cells in rat. OFQ-inducedoutward current was not affected in the presence of TTXand Ca2q-free solution, indicating a direct postsynapticresponse of the neurons. The OFQ-induced current re-

Ž .versed at y99.06"3.80 mV 3 mM , which were quiteclosed to the Kq equilibrium potential as calculated by the

( )T. Amano et al.rBrain Research 853 2000 269–274 273

Ž . qNernst equation E sy96.08 mV, 3 mM for K in ourk

standard solution. This suggests that the OFQ-inducedcurrent was Kq current. Similar results have been previ-

w xously reported in mice hippocampus CA3 neurons 9 .In the hippocampus, OFQ acts as an inhibitory modula-

tor regulating synaptic transmission and synaptic plasticity.The loss of the OFQ receptor results in a gain-of-functionmutation in both the memory process and the long-termpotentiation mechanism in CA1, perhaps as a result ofaltered intracellular signal transduction systems in neuronsw x10 . It has been suggested that activation of OFQ recep-tors may play an important role in synaptic plasticity

w xinvolved in learning and memory 20 . OFQ induces hy-perpolarizing and outward currents via inward-rectifier Kq

channels in mouse hippocampal pyramidal cells, suggest-ing that OFQ receptor couples with the G-protein-activated

q w xK channel 9 .In our study, OFQ-induce outward current was antago-

w 1 Ž . 2 xnized by pre-application of Phe c CH -NH Gly Noci-2Ž .ceptin 1–13 NH , but was not antagonized by pre-applica-2

tion of the other OFQ antagonist on nocistatin.w 1 Ž . 2 x Ž .Phe c CH -NH Gly Nociceptin 1–13 NH has been2 2

proposed as a selective antagonist of OFQ receptor in thew xguinea pig ileum and mouse vas deferens 7 . However,

w 1 Ž . 2 xothers demonstrated that Phe c CH -NH Gly Nocicep-2Ž .tin 1–13 NH acts as a potent agonist in rat spinal cord in2

w xvivo 3,19 and recombinant Chinese hamster ovary cellsw xexpressing the human OFQ receptor in vitro 2 . The

contrary behavior of the pseudopeptide may suggest thatdifferent OFQ receptor types are being addressed in the

w 1 Ždifferent tissues. We have shown that Phe c CH -2. 2 x Ž .NH Gly Nociceptin 1–13 NH inhibited OFQ-induced2

outward current without affecting holding currents at y60w 1 ŽmV. This supports the concepts that Phe c CH -2

. 2 x Ž .NH Gly Nociceptin 1–13 NH acts as a selective antag-2

onist of OFQ receptor in the rat hippocampus pyramidalneurons.

In contrast, we have failed to demonstrate that anotherw xputative OFQ antagonist, nocistatin 13 , inhibited OFQ-in-

duced outward current in rat hippocampus CA3 neurons. Ithas been demonstrated that nocistatin is a new biologicallyactive peptide produced from the same precursor as OFQ.Nocistatin blocks OFQ-induced allodynia and hyperalge-sia, and attenuates pain evoked by prostaglandin E2 invivo. Nocistatin binds to the membrane of mouse brain and

w xof spinal cord with high affinity in vitro 13 . However,w3 xnocistatin does not displace bound H OFQ and does not

prevent the inhibition by OFQ of forskolin-induced accu-mulation of cyclic AMP in Chinese hamster ovary cells

w xtransfected with the OFQ receptor 13 . This suggests thatantagonistic effect of nocistatin on OFQ-induced allodyniaand hyperalgesia is not due to OFQ receptor-mediatedevent. Present study also suggests that nocistatin is not aOFQ receptor antagonist in the rat hippocampus.

It has recently been demonstrated that mice lackingOFQ receptor possess greater learning ability and have

w xbetter memory than control mice 10 . Moreover, OFQreceptor-deficient mice showed larger long-term potentia-tion in the hippocampal CA1 region than control mice.These results show that the loss of the OFQ receptorresults in a gain of function mutation in both the memoryprocess and the long-term potentiation mechanism in CA1w x w 1 Ž . 2 x10 . We have shown that Phe c CH -NH Gly Noci-2

Ž .ceptin 1–13 NH inhibits OFQ-induced outward current2

in hippocampus CA3 neurons in rats. It is conceivable thatw 1 Ž . 2 x Ž .in the future, Phe c CH -NH Gly Nociceptin 1–13 NH2 2

may be useful to treat the patients in dementia.

Acknowledgements

This study was supported in part by the fund fromŽ .Daiichi Pharmaceutical Tokyo, Japan and the National

Institute of Diabetes and Digestive and Kidney DiseasesŽ .RO1 DK55808-01 .

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