μ-Opioid receptor activation decreases N-type Ca2+ current in magnocellular neurons of the rat basal forebrain

Brain Research 75s ( 1997) I 1% 1%

Researchreport

I-L-Opioidrece tor activation decreasesN-type asal forebrai neurons of t

Abstract

Opioidmodulation of calcium currents was studied in acutely dissociatedrat basal forebrain neurons using the whole cell patch-clamp recording technique. The p-opioid receptor agonist DAGO reversibly suppressedhigh-voltage activated calcium currents arid slowed tbeil iate of activation, while neither is- nor K-opioid receptor agonists were effective in modifying calcitim current in these neurons. The inhibitory effect of DAGO on calcium current was abolished following irreversible blockade of N-type calcium channels by w-conotoxin GVIA, whereas DAGO-induced inhibitory responseswere not affected following blockade of L-type calcium channels by nifedipine. These findings indicate that kopioid receptorsare negatively coupled to N-type calcium channelson the postsynaptic membrane of basal forebrain neurons. Calcium currents recorded from a significant number of large. p-opioid sensitive neurons were also suppressedby muscarinic receptor activation, while smaller, p-opioid sensitive neurons were not sensitive to muscarinic receptor activation. Thus, the present data demonstratethat voltage-activatedcalcium influx in several subpopulations of basal forebrain neurons WI he regulated by p-opioid receptoractivation. These results suggestthat p-opioid regulation of calcium current may be an important functional mccbanism in regulating neuronal excitability and synaptic transmission in the basal forebrain.

activation of p-opioid

1. Introductian bridia&m

rend bincling studies [ 17,211and in situ hyanulysis for cxpressim of popicdid receptor

mRNA [10,20,X3] reveal a moderately high density of popioid receptorswithitl several chslint;rgic basal forebrain nuclei, including the medial septum and diagonal band, These uata suggest that popioid receptors might regulate the function of cholinergic neurons within the bawl forebrain. Nevertheless,it is unclear whether these spioid receptorsare localized presynapticallyto the cholinergic projection neurons,possibly acting indirectly to modify their excitability, or whether they intluence the activity of cholinergic neuronsdirectly via a postsynapticaction, Although there has been no prior examination of the cellular responsesof basal forebraincholinergic neuronsto apioid action, results from release studies have provided strong evidencethat opioids regulate the function of these neurons. For example, Lapchak et al. [ 181 reported that

l

Corresponding author. Fax: + I (3 13) 936-8813: E-mail:

[email protected] ~8%3~/97/$17.00

Ccqyripht Q 1997 Elscvicr Science B.V.

PI/ s0006-8993~97!002064

rcccptor~, pre~mably

located ol1

nerve endings of cholincrgic projection neurons, inhibited the depolarizationcvokcd releaseof ncctylcholinc from rat cortical and hippocampalslices. In addition, Moroni et al. [26] have shown that intmseptal administration of PC-opioid receptor agonists reduces acetylcholine turnover in the hippocampus, presumablyvia an inhibitory action exerted at the level of the cell bodies of&olinergic neurons. The mechanism(s) by which opioids phoducethese inhibitory effects remains in question. However, a substantialamount of evidence obtained primarily from recordings from the cell body suggests that opioids could inhibit neurotransmitter release from the presynaptic terminal by reducing the depolarization-evokedinflux of calcium ions (Ca” ) through voltage-sensitiveCa” channels. Suppressionof high-voltage activated (HVA) Ca” currents by p-opioid receptoractivation has been well characterized in sensoryneurons[32,35,36] and neuron&like cell lines [25,37]. It has only recently been demonstratedthat p-opioid receptor activation can also suppressHVA Ca”’ currents in central neurons. Activation of postsynaptic CL-opioidreceptorssuppressesCa’+ current in neurons of

All rightsreserved.

onto the stage of 391

basalis of Mcymrt. Hilti\

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am.1 portions pL9llitlUS

01’Oh.! subst~m~iu iimomidi~bccfcd fiXHI 400 piI1 f IOOC~ 0, I. IOW (‘a’ + CXlra-

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coro9x.1lslices in oxyg iltd cellular solution (in m a. I CaCl 2, 10 HEPES, mosm) with kynurcnic acid (0.5 mM) added. The tissue fragments were transfcrrcd to oxygcnnted W S % 0,/S% CO,) Ca”‘“-free Hank’s Balanced Salt Solutioi~ (HEX%) supplementedwith 2 1 m NaHCO,, and enzymatically treated with trypsin (type I, 1 mg/ml) at 37°C for 30-45 m in. washed, and mechanically dissociatedusing several passagesthrough a fire-polished Pasteur pipette. After cellular debris had been allowed to settle for several m inutes,dissociatedneuronsin the supernatantwere plated onto either poly-L-lysine-coated35 m m culture dishes or similar dishesthat had been modified to include a IO-mm cell well. Neurons were maintained at 37°C in an atmosphereof 5% CO, for approximately I h, after which I m l growth media of the following composition was added to each dish: Dulbecco’s Modified Eagle’s Medium (with L-glutamine,400/J, Ham F- I2 (40%). heat-inactivatedfetal calf serum (IO%), horse serum ( IO%), D-glucose(0.35%), Penicillin/Streptomycin ( I %) and NGF ( 100 @ /ml). Neurons that had well-rounded cell bodies ( 16-35 pm)

rt’uts by using ;in on-line ~41911~” experiments, followir ic)ici-nlc,tdulation

01’ Ca” ’ current,

rmicm pr~:kkx3l.

I91

tim of‘ tc sting ti19 ? + cv;1\ 200 p.

~~~ll~l~i~i~t~r~~~ tc! block ;iII C’u’’ cUr9-c97th a9lJ lo c’txif‘y (:hat 110 outward current remained at a potentiaI that elicited maximal Ca”+ currents. Ci1'+ current ~~i~~I9litudes were measured iso~l~r~~~~i~~~lly near the peak of the response (usually 8- I2 ins following current activation) for each neuron before, during and after drug application. To COIItrol for potential confounding effects of Ca’.’ current rundown on the quantification of agonist-induced rcsponses,time-dependentdeclines in the current recorded over the course of an experiment were accountedfor by using a second-orderpolynomial equationto fit amplitudes before drug perfusionand following drug washout.ControI amplitudeswcrc interpolatedfrom the fit equationover the time period of drug perfusion. averagedand comparedto the actual averaged Ca’+ current amplitudes measured during drug perfusion. Drug-inducedmodificationsin peak Ca’+ current amplitudes are expressedas a 5%changefrom control unIcssotherwise stated. All data were analyzedfor statistical significance using an unpaired Student’s t-test. unless otherwise indicated, and expressed as meank S.E.M. in the text.

~rwedures for the storage and preparation of the Ca’’ channel blockers, w-conotoxin-GVIA (GVIA) and nifedipine (NIF), the L-type Ca’+ channel agonist, BAY K 8644, and the opioid antagonist, naloxone, have been previously described [32]. The CL-opioid receptor agonist [DAla?,MePhe’,Gly-ol”]-enkephalin (DAGO, Peninsula Laboratories) and the Sopioid receptor agonist [D-Pen’>’ l-enkephalin (DPDPE, Peninsula Laboratories) were prepared as 10 mM stock solutions in sterile water, apportioned into IO-p1 aliquots, lyophilized and stored at - 20°C. The K,-opioid receptor agonist U69,593 (Research Biochemical Intl.) was dissolved in 95% ethanol to a stock concentration of 10 mM. The muscarinic receptor agonist, carbachol (Sigma), was dissolved in distilled water to 200 mM. Stock solutions were diluted prior to the experiment with the standardextracellular solution to the desired concentrations. Drugs were applied by local superfusion of the individual neuron under study using a modified U-tube delivery system[24].

3. Results

Neurons isolated from the basal forebrain could be scparatcd into three general groups based on mean somal diameter; (i) small neurons ( 12- 15 pm), (ii) medium-sized neurons (W-23 em), and (iii) large neurons (24-35 pm). Voltage-clamp recordings were obtained from 66 meditmlto large-sized neurons. Maximally activated inward Ca”’ currents ( Ic,,) recorded from these neurons dcmonstri~tcd fast activation kinetics with an initial transient component followed by a longer sustainedcomponent (Fig. l), with a tht@shaldof activation z -40 mV. These characteristics are similar to what has been reported previously for the high voltage-activated /ca in rat basal foreb:brdinneurons [ I]. Only 18% of the neurons that were examined (12= 12) demonstrated low voltage-activated Icu, with a threshold of activation less than -40 mV. These neurons could not bc distinguished by size from those without 101~voltageactivated currents.

The effects of activation of CL-,& or K-opioid receptors on &- were examined by evoking currents at 30 s intervals using 100 ms depolarizing stepsof - 10 to + 10 mV from a holding potential of -80 mV before, during application and after washout of opioid receptor type selective agonists. In 27 of 37 neurons, the p-opioid receptor agonist DAGO (1 FM) reversibly suppressedthe peak amplitude Of I&, by an average of 16.4 f 1.8% (Fig. 1A). The inhibitor-yeffect of DAGQ on &, was not representedby a

100ms

Fig. I. DAGO suppresses high-voltage activated calcium current ( I,.,, 1 in dissociated basal forebrain neurons. A ( rrl~p~r IUX~.S): whole-cell voltage-clamp records of I,., elicited by stepping to 0 mV from b’,, = - 80 mV for a duration of I(H) m\. Local application of’ DA60 ( 1 PM) produced a reversible suppression of Ic;, . A ( bo~nnt tmce): current trace represents the amount of I,, inhibitiLl by DAGO. calculated by subtraction of current record obtained in the presence of DAGO (2) from that obtained before opioid application (I). Note that the peak 1,,, current recorded within the first 8 ms after the start of the depolarizing pulse wits inhibited to a greater extent by DACO than was the sustained current component mcnsured at 100 III\ (times indicated by dotted lines in A). B: the preferential inhibitory effect of DAGO on the trim\ient component of whole-cell Gil-” current is clearly illustrated in current records evohcd with longer (S(H) a14 test pulse\. C: log-concentriltioti-rehpon~e relationship for inhibition of peak I,., amplitudes by DAGO. E,,,,,, = X’.X*ii, EC,,, = 0.7 PM. /I = I-‘).

uniform reduction in current amplitude tlrroughoul the duration of the dqwlarir.in~ pulse. Rather, the peak amplitude of Ici, was reduced to a greater extent than was the current measured near the end of a 100 ms depolarizing step (Fig. 1A , ). As a result, the ratio of the amplitudes of the peak (8 ms) and late-sustained( 100 ms) components of Licitwas significantly reduced by l-3 /ILM DAGO from 5.21 + 0.02 to 1.13 3-0.03 (12==40; P
20 m8

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CL-opioid rcccptorx. A: numhcred records of I, ,, were cv~~hed by lot) Ill\ Fig. 2. DAGO-induced suppression of /,;, is due to activation of postsynaptic voltuge steps to 0 niV from V,, = - X0 mV and inensured ill the corrchponding pointh indicated in the plot of peak current amplitude versus IlIne. D A G O ( 1 PM) reversibly suppressed I,,,, and induced a slowing of current aztivution. The p-opioid receptor antagonist IlilitlXOIle ( I PM) ~OIK? did not Ill~~dI1j if,.,. but reversibly blocked the DAGO-induced inhibition of current. B: records (left to right) are subtraction cum111 trace\ of DAGO-sensitive I,., (WC f;@ 1 ) C: time course of the experiment illustratc~ iIlilihlli~~Il recorded under control conditions. in the presence of naloxone. und following washout of ~laloxo~lc. in I,,, produced

by DAGO

before. during

administration

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washout

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DAGO-sensitive current

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45

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Time (min) Fig. 3. RAG0 selectively inhibits N-type Ca” channels. A: records (left to right’ of 1,, measured before and after local appllication of DAGO ( I gM) under control conditions, in the presence of the L-type Ca’+ channel blocker nifedipine (NIF; IO PM), and in the presence of NIF itnd the N-type Gil’+ channel blocker GVIA (1 PM). B: subtracted current records (see Fig. I) show the amount of current inhibited by DAGO under the corresponding conditions in A. C: plot of peak 1,, versus time after patch rupture illustrates inhibition in I,.,, by DAGO administered alone. in the presence of NIF (hatched bar) and after exposure to GVIA (indicated by downwitrd arrow). D: graphical comparison of the inhibitory effect of DACO on I(.., measured in the absence and presence of selective N-type (GVIA) and L-type (NIF) CiI’ ’ channel blockcrh; (I) DAGO (3 PM) inhibited /,.,, by 18.0 k 7.O’i hcforc and 2.6 f 1.4% after exposure to GVIA ( ’ P < 0.05); (2) DAGO inhibited I(.,, by 3.8 f 4.9% before irnd 16.0 k 3.7 ilfter cxposurc IO NIF (n.s.1.

sion of neuronswith NIF ( IO PM) for 30-60 s rwcrsibly blocked fc,, by 23.4 3,Oc/c(II = IS), whercns application of GVIA (1-3 pM t’or IO s wultcd in an irrcvcrsiblo blockirdcat’ I,, that nvcraged Y&S 3.0% (II = 18). The effect of co-upplkntion of both NIF nnd CVlA on !c[, wus akiitive, nducing current amplitudeby 48.7 f 3.2% (1) =5

3 neurons, QAGO inhibited the control /<.,, by an ;kvcriigc of 10.7 & 4.3% (Fig. 4A, inset), and the inhibitory cf’l’txts A Control medium

afttsrGVIA

181,while the residualcurrent that remainedin the presence af both antagonists was entirely blocked by applica-

tion of Cd’” (200 FM). Blockadeof L-type channelsby NIF did not significantlyreduceCL-opioidinhibition of Ica (Fig. 3). In tests on 5 neurons,the amount of I,, suppressedby DAGO (1 &Ml after blockadeof L-type current with NIT:(16.9 f 3.7%) was not signiticantlydifferent from the reductionin current producedby the p-opioid agonistprior to administrationof the dihydropyridine(23.8 f 4.9%). In contrast,after establishmentof an irreversible block of N-type current by applicationof GVIA (I PM), DAGO did not modify /c, (2.0 f 0.9%, n.s.), whereas DAGO suppressedlcil by 16.7f 3.5% before application of GVIA in the same neurons (II = 9: Fig, 3 and Fig. 4A, right traces).The effects of administrationof GVIA and NIF on DAGO-inducedinhibition of Ica are compared graphicallyin Fig. 3D. To further test whether popioid receptorsare coupled to L-type channels,we studiedthe effect of DAGO on the

BAY K

4

DAGO

Control

I02nA 3ms

3ms ‘kontrol

&nA I 10ms

Fig. 4. BAG0 does not modify GVIA-insensitive and BAY K X644-enhunted current. A ( imet 1: records of i(., evoked by 200 ms voltuge steps to + IO mV from V,, = -80 mV demonstrate a I&5% suppression of current by DAGO (I PM). A and B: records of I,.;, from the same neuron evoked with 30 ms voltage pulses at expanded time scale to show the tail currents. A: application of GVIA t I PM) reduced the amplitude of &., and peuk rail current by 46%. after which reapplication of DAGO was without effect. B: administration of BAY K 8644 (IO PM). subsequent to application of GVIA, induced a slow phase in the tail current. Administration of DAGO in the continuous presence of BAY K did not modify the BAY K enhanced tail current.

neurons, many

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defined 19y the ability crf‘ the ~i~~~s~~ri~~i~ I’txuptot curb;k4iol to SLI19rchh I‘.,,. Thd’ records in Fig, 5 iirc from three differc~~tbasal forebruin neurons that represent the three distinct profiles of Ici, inhibhion to p-opioid and muscarinic receptor activation that were found: I(., was either (I ) FL-opioid sensitive, but not cholinoceptivc ( SA); (2) g-opioid sensitive and cholinoceptive (Fig. or (3) cholinoceptive, but not p-opioid sensitive Wig. SC). Nineteen out of 22 neurons tested were cholinoceptive, as determined by the ability of carbachol (IO ELM) to suprcms

were

agonist

ile 1I cholinoccpti (one neuron wits not tcs Interestingly, the mount of current su in cholinoceptivc neurons C 17.3 t- 3.4% age. different from that suppressedby that were non-cholinoceptive (22.5 &I 8. data suggest the possibility that more’than one ccli type

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* CSA (cross-sectional area) = T. ((1,,,,,[,/2) . (d,,,,,/Z). where d is the diameter of the short or long axih of the SOllli~. h In parentheses, are numbers (if different from II) of neurons tested for sensitivity to DAGO at a concentration of I pM. ’ P < 0.05 for the two values compared within the specified column: all other comparisons are not significantly different.

.-s

cua-rents

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~~~~~~~~~~ [I] Allen, T.G.J. inld Brown, D.A., The whole-cell CulchmI current in ilClltf2ly diswciattxl llIil~lIWXllllliW cholincrpic tsilSill forebruin IlCllroncs Of the rilt. J. f’h~.\iol.. 400 (lOO3) 01 - I 16. [2] All011. T.C.J. and Brwvn. D.A.. M z muhrarinic r~ccl)tc)r-alcdi;ItL”d cholinorpic IXMII inhibition of the Ca” ’ current in lXt lllil~llOC~llUlilr forebrain neurones. J. f’~~,wiol., 466 ( 1993) l73- I X9. [3] Arenas, E.. Albcrch. J., Sanchez Arroyos, R. and Mars& J., Effect of opioids OII acetylcholine release evoked by K’ or glutamic acid from rut noostriatal slices, Bruirr Rex, 523 ( I9901 5 I -56. [4] Bean, BP., Neurotransmitter inhibition of neuronid cidciul~~ currents by changes in chitnncl VOIU~C dcpendcnce. NU~UW, ,740 ( IYXY) IS-1%. [S] Brasheilr, H.R.. Zaborstky, L. iuld Heimer, L.. Distribution of GABAergic and cholinergic neurons in the rat diiIeOni\l bitnd. NUIrcrst~ierfcv.I7 ( IYXh) 430-45 I. [S] Buckley, N.J.. BWIW, T.1. and Brann. M.R.. l.()cali/inlr)n 01’iI family of muscarinic rcccptor mRNAs in rat brain. J. Nwrosc~.. X (I’)881 4646-4652. [7] D&our, A.H.. Lipscombe. D. and Tsicn. R.W., Multiple mode\ oi N-type culcium channel activity distinguished by diffwnccs in gating kinetics, .I. ftkrrrosci.. I3 ( I993) I K I - IO4. [8j Dunlap, K.. Luebke. J.I. and Turner, T.J.. Exocytotic Cu’ ‘ chimnels in mammuliun central neurons, Trds NCUIVJ.S& 1X ( 1005) 89 -98. [9] E&en tein, F. and Sol’roniew. h$ ‘I., Identification of central hlin-

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ai~ti\gonists on strintal acetylcholine and dopamine release. Brain Rex Bull.. 29 (1992) 369-373. [35] Schroeder. J.E.. Fischbach. .S.. Zheng. D. and McCleskey. E.W., Activation of EL-opioid receptors inhibits transient high- and lowthreshold Ca’ + currents. but spiieS a slhtilirled cumnt. Nfwrm. 6 (1991) 13-20. [36] Schroeder. J.E. and McCleskey. E.W.. Inhibition of Ca’+ currents by a p-opioid in a defined subset of r;lt senhq IUIOPI~. 9. Netrrawi.. 13 (1993) 867-873. [37] Seward. E.. Hammond, C. and Henderson. G., CL-Opioid-receptormediated inhibition of the N-type calcium-channel current. Pn)c*. R. Sot. Lmd. B. Biol. Sci.. 244 (1991) 129- 135. [38] Shen, K.F. and Grain. S.M., Dual opioid modulation of the action potential duration of mouse dorsal root ganglion neurons in culture, Brcritr Res.. 491 (1989) 227-242. 1391 Soldo. B.L. and Moises. H.C. Demonstration of p-opioid receptor suppression of Ca’+ currents in central neurons, .Sf)c.. Nmrfm’i. Abstc. 21 (1995) IRS1 (Abstract) [40] Stefani. A., Surmcicr, D.J. and Bcrnardi. G.. Opiuid~ Jecrcihse high-voltage aciivated calcium currents in acutely d~ssoci;lted ncostrintal neurons, Bntiu Kt~s.. b4Z t 1994) 339-34.X 1411 Zabonzky. L.. C&en. J., Brashear. H.R. and Hcimer. L., Cholinergic and GABAerpic aft&ems to the olfactory bulb in the rat with special emphasis on the projection neurons in the nucleus of the horizontal limb of the diagonal b;md. J. Ci~rq~. &lrrol.. 243 ( 1986) 488-509.