richard bergeron, claude de montigny and guy debonnel- potentiation of neuronal nmda response...

8/3/2019 Richard Bergeron, Claude de Montigny and Guy Debonnel- Potentiation of Neuronal NMDA Response Induced by D

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The Journal o f Neuroscience, February 1, 1996, 76(3):1193-l 202

Potentiation of Neuronal NMDA Response Induced byDehydroepiandrosterone and its Suppression by Progesterone:Effects Mediated via Sigma ReceptorsRichard Bergeron, Claude de Montigny, and Guy DebonnelDepartment of Psych iatry , Neurobiological Psych iatry Unit, McGill University, Montreal, Quebec, Canada H3A 1A 1

We have shown previous ly that low doses o f selective sigma(cr)-receptor ligands potentiate the excitatory response of py-ramidal neurons to NMDA in the CA, region of the dorsalhippocampus in the rat. Because progesterone competi tivelydisplaces the binding of the ligand N-[3H]allyl-normetazocine(SKF-10,047), the present studies were undertaken to deter-mine in viva the eff ect of neuroactive steroids on NMDA-induced excitation of rat CA, pyramidal neurons. Low doses ofdehydroepiandrosterone (DHEA) potentiated the NMDA re-sponse selectively and dose-dependently. The ef fec t of DHEAwas reversed by the selective v antagonist N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl)-ethylamine monohydro-chloride (NE-100) and by haloperidol, but not by spiperone.Progesterone had no eff ect by itse lf but reversed, at low doses,the potentiation of the NMDA response induced by DHEA as

well as those induced by nonsteroidal u ligands. Neither preg-nenolone nor pregnenolone sulfate had any eff ect on the NMDAresponse-nor did they antagonize the potentiation of theNMDA response induced by DHEA and by nonsteroidal IJligands. A pet-tussis toxin pretreatment, which inactivates G,,,-proteins, abolished the potentiating effect s of DHEA. Ovariec-tomy enhanced the potentiation of the NMDA response by thenonsteroidal CT igand di(2-tolyl)guanidine (DTG). There was areciprocal occlusion of the eff ect s of DHEA and DTG; DTG didnot potentiate the NMDA response further after DHEA, andDHEA did not do so after DTG. These results suggest that someneuroactive steroids modulate the NMDA response via greceptors.

Key words: neurosteroids; hippocampus; electrophysiology;ovariectomy; haloperidol; DTG

Sigma ((7) receptors are present in high density in the CNS(Walker et al., 1990) and in peripheral organs, with a particularlyhigh density in the ovaries (Wolfe et al., 1989). Many psychotropicdrugs such as antipsychotics and antidepressants have high aff ini tyfor v receptors (Su, 1982; Schmidt et al., 1989; Snyder andLargent, 1%); ltzhak and Kassim, 1990; Ferris et al., 1991).Several steroids, progesterone in particular, also bind with highaff ini ty to (7 receptors (Su et al., 1988).

Previous studies have demonstrated that low doses of selective CTligands, such as di(2-tolyl)guanidine (DTG) (Weber et al., 1986),I-benzylspiro[ 1,2,3,4-tetrahydro-aphthalene-1,4-piperidine] (L-687,384)(Middlemiss ct al., 1991; Barnes et al., 1992), (+)N-cyclopropylmethyl-N-methyl-l ,4-diphenyl-1-ethyl-butyl-3-N-I-ylamine hydrochloride (JO-1784) (Roman et al., 19Y0), and (+)pentazocine (Steinfels et al., 1988)select ively potentiate the response of rat CA, dorsal hippocampus py-ramidal neurons to microiontophoretic applications of NMDA (Monnetet al., 1990, 1992; Martin et al., 1992; Walker and Hunter, 1994). Thiseffec t is reversed by other u ligands such as haloperidol, (+)3-hydroxyphenyl-N-i-( 1 propyl)piperidine [also known as (+)3-PPP],and cr-(4-fluorc~phenyl)-4-(5-flu~~ro-2-pyrimidinyl)-l-piper~ine butanol(BMY-14802) (Largent et al., 1984; Tam and Cook, 1984; Taylor and

Rrceivcd Aug. 8, lYY5 ; rcviscd Oct. IO, IY YS : accepted Nov. I I, 199.5.This work ws supported by the Medical Rcscarch Counci of Canada, the Royal

Victoria Hospital Research Inst itute , and the Fends de la Recherche en SantC duOu&ec (FRSO). G.D. received II scholarshiD from the FRSQ, and R.B. received afellowship from the FRSO. We thank T.Vo for computer program ming andstatistical ;malyses. C. Bouchard for illustrations, and L. Martin for secrctxiala\\i\tancc.

Corre\pondcnce should he addressed to Richard B ergcro n, Departm ent of Psy-chiatry. Neurohmlogical Psychiatry Um t, McGill UnivcrGty, IO.13 Pint Avenue West.Montreal. Quchec, C;mada H3A I Al.Copyright 6: IY YO Society for Ncuroscicnce 0270-6474/9(,/lhl lY3-lO$OS.OO /O

Dekleva, 1987), but not by spiperone, which has a binding profile similarto that o f haloperidol except for its low affin ity for v receptors (Monnetet al., 1990, 19Y2). We also have tested recently the novel and veryselective (T ligand N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl)-ethylamine monohydrochloride (NE-100) (Okuyama et al., 1993). Itproved extremely potent in blocking or reversing the potentiation of theNMDA response induced by v ligands such as DTG (Debonnel et al.,1995a). Therefore, g ligands acting like DTG have been denoted ten-tatively cr agonists, and (r ligands acting like haloperidol have beendenoted antagonists (Monnet et al., 1990). These data and severalother reports from other laboratories using biochemical, neuroendocri-nological, and behavioral models suggest the existence of a functionalinteraction between (T and NMDA receptors, although the exact mech-anism is not understood ful ly (for review, see Dcbonncl, lYY3).

There are several types of (T receptors; those classified as (r, andcr, have been characterized most extensively (Quirion et al., 1992).The most commonly used g ligands, including haloperidol andDTG, do not discriminate between CT,and (r7 receptors (Quirionet al., 1992), whereas drugs such as (+)pentazocine, JO-1784,L-687,384, N-allyl-normetazocine [( +)SKF-100471, and NE-100are selective for g, receptors (Quirion et al., 1992; Chaki et al.,1994). However, recent data obtained in our laboratory haveshown that pertussis toxin (PTX) pretreatment abolishes thepotentiation induced by JO-1784, but not that induced by (+)pen-tazocine (Monnet et al., 1994), which suggests that these two (rligands ac t on different u-receptor subtypes. Moreover, the injec-tion of colchicine in the dentate gyrus, which destroys the mossyfiber system (a major af ference to CA, pyramidal neurons), abol-ishes the potentiation of the NMDA response induced by DTGand JO-1784, but not that of (+)pentazocine, suggesting that thesubtype of u receptors on which DTG and JO-1784 are acting is


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1194 J. Neuros ci., February 1. 1996, 76(3):1193-1202

located prcsynapt ically on the mossyfiber terminal, whereas thesubtype of u receptors on which (+)pentazocine is acting islocated postsynaptically (Debonnel et al., 199%).

Several reports have shown that progesterone acts as a competitiveinhibitor of [3H](+)SKF-10047 or [H]haloperidol (Su et al., 1988;iLk&nn et al., 19Y4; Yamada et al., 1994; Ramamoorthy et al., 1995)at (r receptors and have prompted us to assess the potential agonisticor antagonistic properties of progesterone, dehydroepiandrosterone(DHEA), and other neuroactive steroids (Paul and Purdy, 1992) inan bz r,it~ clectrophysiological paradigm.MATERIALS AND METHODSThe experiments were carried out i/z t,il~ in the CA, region of the ratdorsal hippocampus, in which the responsiveness of pyramidal neurons tomicroiontophorctic applications of NMDA, quisqualate (QLJIS), andacetylcholine (ACh) was assessed using extraccllular unitary recordings.

P~e~~amtiorr qf rr/~ir?ials. Male Sprague-Dawlcy rats weigh ing 200-250gm were ohtainc d from Charles River Laboratories (St. Constan t, Que-bec. Canada). Female rats of the same weight wcrc obtained at day 1 or.? of the menstrua l cycle or 2 weeks after ovariectomy (OVX). Rats werehoused three to four per cage with free acce ss to food and water. Theywere mainta ined at consta nt temperature (25C) under a I2 hril2 hrlight/dark cycle.PTX~ t~ecrt,,le,ft. The pretreatment with PT X (I pg in 2 /.LI of physi-ological saline; Sigma , S t. Louis, MO) consisted of lowering the tip of a5 ~1 Hamilton syringe unilaterally into the right dorsal hippocampus ofchlora l hydrate-ancsthctizcd rats (anterior, 4.5 mm; lateral, 4 mm; anddorsal, 4 mm) according to the atlas of Paxinos and Watson (1986). PTXwas injcctcd slowly over a period of5 min. Control rats were injected withan equal volume ofphy\iological saline under the same conditions. In i~ir~oclectrophysiological cxperimcnts were carried out S-7 d later.Drir~,s, The following substances were used: DTG (Aldrich, Milwaukee,WI); 50-1784, a gift from J. Junien (Institut de Rechcrche Jouvcinal,Fresncs, France); L-687,384 , a gift from L. Iversen (Merck-Sharp andDohmc , Tyle r Park, UK); NMDA and ACh (Sigma): QUIS (T ocrisNeuramin, Buckhurst Hill , Essex, UK): halop eridol (McNeil Lahorato-ries, Stoulfv ille, Ontario, Canada): spipero nc, (+)pcntazocine, progcster-one. prcgnenolonc, prcgnenolone-sulfate, and DHEA (Research Bio-che mica ls, Natick. MA); NE-100, a gift from S. Okuyama (Taish oPharmaceutical. Ohmiya, Japan).

Prc~,clrtr/iorr of irric.r.o~>i/~e /lc.s. Microion tophorctic app licatio ns and cx-tracellula r unitary recordings were performed with S-barrel glass micropi-pettcs prepared in a conventional manner (Haigler and Aghajanian,1974). Three of the side barrels used for microiontophorcsis contained

(in mM): NMDA IO (in NaCI 20(l), pH 8, QUIS I.5 (in NaCl 4 00), pH X,and ACh 20 (in NaCI 200), pH 4. The remaining side barrel, used forautom atic current balan cing, and the central barrel, use d for extracellularunitary recording, were lill cd with 2 M NaCI saturated with Fast Green(FCF; Aldrich).

Rwordiq.t J?ot11 CA., rlor:wl hip,uocanyx~s pynn~ iclul NCLIK~ IIS. For theelectro physi ologic al experiments, rats were ancsthe tizcd with urethane ( I.25g/kg, ip.) and mounted in a stcreotaxic app aratus. Body tcmpcrature was

Bergeron et al. l Neuroactive Steroids and o Receptors

maintain ed at 37C throughout the experiment. After removal of the duramater, the micropipcttc was lowered into the CA, region of the dorsalhippoc ampu s (lateral, +4.2 mm; anterior, +4.2 mm from lambda : dorsal.-3.5 to -4.5 mm from the cortic al surface). Pyramidal neurons wcrcidentifi ed by their long-duration (11.X-1.5 mscc ) and large-amp litude (0.5-2mV) action po tentials and by the presence of characteristic complex spikedischarges alternating with simple spike activity (Kandcl and Spencer. lOhI).

Neuronal firing activity was monitored via an oscillosco pe after signalmagnification by a high-input impedance amplifier. Action potentials weredetected by a differential amplitude discriminator generating square pulses,which were stored on-line and forwarded to a paper chart rccordcr gcner-sting integrated firing-rate histograms. Alternatc microiontophorctic appli-catio ns of SO set of each excitatory subs tance (NMDA. QUIS, and ACh)separated by SO set retention periods wcrc carried out continu ously duringthe duration of the recording. The duration of the microio ntopho rcticapplications and the intensity of the currents used also wcrc stored on-line.The elfccts of their applications on pyramidal neuron firing activity wereexpressed as the number of spikes generated per nanocoulomb (nC: I nC isthe charge generated by 1 nA applied for 1 set).

After a neuron was isola ted, it was recorded for a period of at least 20min before any drug was injected. The recording was stored on-linewithout interruption for the duration of the experiment. Five to sixapplications of each excitatory substance (NMDA. QUIS. and ACh) wcrccarried out before the drugs studied wcrc injcctc d or applied microion-tophoretically. The effects of drugs studied appcarcd within a period of 10min after their intravenous injection. The calculations wcrc carried outwhen the maximal effect of the drug was achieved (within the first 20min). At the end of the experiment, a -27 FA current was passe d throughthe central barrel for 20 min to depo sit Fast Green FCF for subs cquc nthistologica l verification of recording sites.

Esl,crinrerzral se+s. Neuroactive steroids wcrc prcparcd i n ;I solution of40% polyethylene glycol. The (r ligands were prepared in physiologicalsaline, and they were adm inistered via a lateral tail vein. In all cxpcri-mental series, only one dose of each drug was administered to one ratwhile recording from one neuron.

In the first series of experiments, several dose s of each drug wc rc tested togenerate dose-response ctnvcs. Progesterone, DHEA, prcgnenolonc, andpregnenolone sulfate w crc tested at doses ranging from I Kg to 2 mgikg. Inthe secon d series , progesterone (20 pg/kg, iv.) was tested as an antagonist ofthe potcntiation of the NMDA response induced by the previous adminis-tration of a low dose of DHEA or of the following nonstcroid rr ligands:DTG I pg/kg, i.v.; L-(,X7,384 I kg/kg, iv.; JO-17X4 5 &kg, iv.; (+)penta-zocine IO &kg, iv. In the third s eries, rats were pretrcatcd with 21 localinjection of PTX to assess the possible involvement of (r rcccptors coupledwith G,,,,-proteins in the potentiatio n of the neuronat respons e to NMDA bvneuroactive steroids . In the fourth series , we have examined the pos sibili ty 01a reciprocal occlusio n of the cffccts of DTG and DIIEA. In the final scrics.the degree of the potcntia tion of the NMDA response induc ed by DTG (I&kg, iv.) was measured in male rats, in female rats at days I and .i of themenstrua l cycle, and in OVX rats I4 d after the surgery.

Crrk~irkrtions. The computer calcu lated the clfec t of each 50 SK micro-iontophoretic application of an excitatory substance as the total numbctof spikes generatcd/nC. Each value was calculated by the computer as themean of the effect of three consecu tive applications of the same excitatory

F@rre 1. A. Intcgratcd tiring-rate histogram of a CA, dorsal hippocampus pyramidal neuron illustrating the effects of microiontophorctic applicationsof NMDA and QUlS before and after the injec tion of DHEA (crr~ow al /cft) and after the subseq uent admin istration of halope ridol (~~~oM.(I/ rig/i/). In thisand the subsequent integrated tiring-rate histograms, bars indicate the duration of applications for which currents arc given (in nA), and o,>c,i circksreprcscnt an interruption of the illustr ation of the continu ous recording. R, Dose-respon se curve of the cl& t of the intravenous admin istration of DHEAon the neuronal activation of CA, dorsal hippocampus pyramidal neurons induced by microiontophoretic applications of NMDA. Each c/or rcprcscntsthe effect of one dose of the drug administered to one rat while recording from one neuron in this and subsequent doseresponse curves. T he clfcct wasasse ssed by determining the ratio (N,:N,) of the number of spikes gcneratcd per nC of NMDA before (N,) and after (NJ) the inje ction of the drug. C,Responsiveness. cxprcssed as the number of spikes generated per nC (mean 2 SEM), of CA, dorsal hippocam pus neurons to microiontophorcticappl icatio ns of NMDA before (o/jen cohmm s) and after (grcry cohrm/r.s) the admin istration of DHEA and after the subs cquc nt admin istration ofhalop cridol (&I& cohmzr~.s). The rrrr&er- within the ope,r colrrnirzs ind icate s the number of neurons tcstc d (I neuron/rat in this and subs cquc nt bar charthistograms). D, Rcsponsivencss, expressed as the number of spikes generated per nC (mean t SEM), of CA, dorsal hippocampus neurons tomicroion tophore tic app licatio ns of NMDA before (opefz cohrmns) and after (fiNy co/rrrn,zs) the admin istration of DHEA and after the subs cquc ntadmin istration of spipero nc ((lurk cohrnm~). The nwnher- within the o,lerl colltr~zn indic ates the number of neurons tested (1 neuron/rat in this andsubsequent bar chart histograms). E, Responsiveness, expressed as the number of spikes gcncrated per nC (mean ? SEM), of CA, dorsal hippocampusneurons to microio ntopho rctic app licatio ns of NMDA before (oven colrtrnns) and after @uy cohrm!z.s) the admin istration of DHEA and after thesubsequent administration of saline (&irk co7m7r7.s). F, Responsiveness, expressed as the number of spikes generated per nC (mean i- SEM). of CA, dorsa lhippoc ampu s neurons to microio ntophore tic app licatio ns of NMDA before (o/le,i co/rrm,z.s) and after (grrry colnnnr,s) the admin istration of DHEA andafter the subsequent administration of the selcctivc (7 antagonist NE-100 (t/t?& coh~~n~i.s). A.s~ri,sks in C-F in dicate 1 < 0.05 using paired Students I test.


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Bergeron et al. . Neuroactive Steroids and CTReceptors J. Neurosci., February 1, 1996, 16(3):1193-1202 1195

AQUIS NMDA

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cl ControlDHEA (250 pg/kg, Lv.)Halop eridol (20 pg/kg, i.v.)

* x

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I NE-100 (25 pglkg, i.v.)


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1196 J. Neurosci., February 1, 1996, 16(3):1193-1202 Bergeron et al. . Neuroactive Steroids and CTReceptors

Figwe 2. A, Integrated firing-rate histogram of a CA,dorsal hippocampus pyramidal neuron illustrating the ef-fect of microiontophoretic applications of NMDA, QU/S,and AC/? before and after the injectio n of progesterone(~770~ (71 I@) and after the subsequent injection of halo-peridol (nrrow ut ri& see legend to Fig. 1). B, Dose-response curve of the effect of the injection of progester-one on the neuronal activation of CA, dorsalhippocampus pyramidal neurons induced by microionto-phoretic applications of NMDA. C, Responsiveness, ex-pressed as the number of spikes g enerated per nC (mean+ SEM), of CA, dorsal hippocampus pyramidal neuronsto microiontophoretic applications of NMDA before(open colum ns) and after (gray cobrmns) the adminis tra-tion of DHEA or progesterone and after the subseq uentadmin istration of either steroid (dark colum ns). Asteriskindicates p < 0.05 using paired Students t test.

A ACh QUIS NMDA9 -5 -11

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Progesterone Halopendol100 pg/kg, i.v. 20 Kg/kg, i.v.

3-

2-

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Dose (pg/ kg, i.v.) of progesterone

substances. The effects of the intravenous administration of the drugsstudied were assessed when the maximal effect was achieved, by deter-minin g the ratio (N?:N,) of the number of spikes generated/nC of each ofthe three excitatory subs tance s ACh, QUIS, and NMDA before (N,) andafter (N,) the injec tion of the drug. The dose-respo nse curves of theeffects of intravenous administration of CT igands were obtained by fittingexperimental data to general logistic equations obtained with the soft-ware Tablecurve (version 3.0, Jandel Scientific, San Rafael, CA).

Sruristical ~7u7IJI~e.s. All results are expressed as mean % SEM of thenumber of spikes gencrated/nC of NMDA, QUIS, or ACh. Statis tical

ControlDHEA (250 pg/kg, i.v.)Progesterone (20 pg/kg, i.v.)

2.0

1.5

1.0

0.5

0

cl ControlProgesterone (20 pg/kg, i.v.)DHEA (250 pglkg, i.v.)

significance was assessed using Students t test with Dunnetts correctionfor multiple comparisons. Covariance analysis was used to compare thedegree of the potentia tion of the NMDA response induc ed by DTG (1Kg/kg, i.v.) in male and in non-OVX and OVX female rats; p 5 0.05 wasconsidered significant.RESULTSAl l recordings were obtained from the stratum pyramidale of thedorsal hippocampal CA, region, as confirmed by histologica l


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ACh QUIS NMDA7 -2 -12

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verifica tion of the Fast Green FCF deposit left at the end of eachexperiment.

The intravenous injection of the vehicle (40% polyethyleneglycol), used for preparing the solutions of neuroactive steroids,affected neither the spontaneous firing ac tiv ity of dorsal hip-pocampus CA, pyramidal neurons nor their response to NMDA,QUIS, and ACh.Effects of DHEAThe intravenous administration of 100 pg/kg DHEA induced atwofold increase of the response of dorsal hippocampal CA3pyramidal neurons to microiontophoretic applications of NMDAwithout affec ting their response to QUIS (Fig. 1A). The eff ect ofDHEA was dose-dependent, and a maximal potentiation wasobtained at a dose of 500 pg/kg, i.v . (Fig. 1B). No additional

1 min

Figure 3. Continuous integrate d tiring-rate histogram of a CA, dorsal hip-pocampus pyramidal neuron illustratingthe effec t of microiontophoretic applica-tions of NMDA, QUIS, and AC% beforeand after the administration of DTG(arrows) and after the administration ofprogesterone. Time scale applies to thethree traces.

potentiation was obtained by increasing the dose of DHEA up to2 mg/kg, i.v. (Fig. 1). This enhancing effec t of DHEA lasted for atleast 40 min. The potentiation of the NMDA response by DHEAwas reversed by haloperidol (20 pg/kg, i.v .; Fig. lC), but not byspiperone (20 kg/kg, i.v .; Fig. 1D) and not by the intravenousadministration of saline (Fig. 1E). Moreover, NE-100, a novelselective v antagonist (Okuyama et al., 1993) at a dose of 25pg/kg, i.v ., also suppressed the potentiation of the NMDA re-sponse induced by DHEA (Fig. 1F).Effects of progesteroneAt doses ranging from 1 to 2000 pg/kg, i.v ., progesterone did notaff ect the neuronal response induced by microiontophoretic ap-plications of NMDA (Fig. 2&?), QUIS, or ACh. However, pro-gesterone, at a dose of 20 kg/kg, i.v ., prevented and reversed the


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1198 J. Neurosci., February 1, 1996, 76(3):1193-1202

0 CUltKiDTG (1 fig/kg. i.v.)

w Progesterone (20 Kg/ kg, iv.)

Cl Control

cl ControlJO-1784 (5 pglkg, i.v.)Progesterone (20 pglkg, i.v.)

D

2.0

1.51.0

0.5

0

0

0 Controlq (+)Pentazocine (10 pg/k g, i.v.) /#f L-687,384 (1 pglkg, i.v.)n Progesterone (20 Kg/ kg, i.v.) H Progesterone (20 Kg/ kg, i.v.)

* *

0 Control Cl Controlfg DTG (1 pglkg, i.v.) q DTG (1 fig/kg, i.v.)I Haloperidol (20 kg/ kg, iv.) Spiperone (20 Kg/ kg, i.v.)

Fgure 4. Responsiveness, expressed as the number of spikes generatedper nC (mean ? SEM), of CA, dorsal hippocampus pyramidal neurons tomicroiontophoretic applications of NMDA before (open columns) andafter (gray columns) the intravenous administration of DTG (A). JO-1784(B), (+)pentazocine (C), and L-687384 (D), and after the subsequentintravenous administration of progesterone (A-D), haloperidol (E), orspiperone (F) (dark columns). Asterisks indicate p < 0.05 using pairedStudents t test.

Bergeron et al. l Neuroactive Steroids and u Receptors

potentiation of the NMDA response by DHEA (250 pg/kg, i .v. ;Fig. 2C). To investigate the possibility that this eff ect of proges-terone could be mediated via c receptors, low doses of theselective u ligands DTG (1 pg/kg, i.v .; Fig. 3), L-687,384 (1 pg/kg,i.v .), JO-1784 (5 pg/kg, i .v. ), and (+)pentazocine (10 pg/kg, i .v. )were administered to naive rats as illustrated for DTG in Figure3. Progesterone (20 pg/kg, i.v .; Fig. 4A-D) reversed the potenti-ation induced by the nonsteroidal v ligands. Haloperidol (20pg/kg, i.v .; Fig. 4E), but not spiperone (20 pg/kg, i.v .; Fig. 4F),also reversed the eff ect of DTG as reported previously (Monnet etal., 1990; Bergeron et al., 1993).Effects of pregnenolone and pregnenolone sulfateThe effects of pregnenolone and pregnenolone sulfate were as-sessed in the same paradigm. At doses ranging from 1 to 2000pg/kg, i.v ., these two neuroactive steroids did not modify NMDA-,QUIS-, or ACh-induced neuronal responses. The eff ica cy of thesetwo neuroactive steroids in reversing the potentiation of theNMDA response induced by a previous administration of DHEA(100 Fg/kg, i.v .), as well as by other nonsteroidal (T agonists, alsowas tested. Pregnenolone and pregnenolone sulfate neither re-versed nor prevented the potentiation of the NMDA responseinduced by DHEA (Fig. 5A,B). Similarly, neither pregnenolonenor pregnenolone sulfate suppressed the potentiation of theNMDA response induced by the nonsteroidal cr agonists DTG (1Fg/kg, i.v .; Fig. .5C,D), L-687,384 (1 pgikg, i.v .), JO-1784 (5 pgikg,i.v .), or (+)pentazocine (10 pgikg, i.v .) (data not shown for thelatter three compounds).Effect of coadministration of DHEA and DTGTo test the possibility that DHEA and nonsteroidal rr ligands bothactivate (T receptors, we have examined the possibility of a recip-rocal occlusion of the eff ects of DHEA and DTG. The microion-tophoretic applications of DTG (20 nA) produced, as observedpreviously (Monnet et al., 1990; Bergeron et al., 1995a), a twofoldincrease in the neuronal response to NMDA. The injection ofDHEA at 200 pg/kg, i.v ., failed to elicit an increase in NMDAresponse. Conversely, the intravenous administration of DHEA at200 pg/kg also induced a threefold increase in the neuronalresponse to NMDA, and the subsequent microiontophoretic ap-plications of DTG (20 nA) failed to elicit an increase in NMDAresponse (Fig. 6).Effect of PTX pretreatment on the potentiation inducedby DHEAPTX, which inactivates G,,-proteins via ADP ribosylation, wasused to assess the possible involvement of these proteins in themodulation of NMDA-induced neuronal activation in the CA,region of the rat dorsal hippocampus by DHEA. The in viva PTXpretreatment affected neither the spontaneous firing act ivi ty ofCA, pyramidal neurons nor their responsiveness to NMDA,QUIS, or ACh, which is in agreement with previous data (Monnetet al., 1994). DHEA, at a dose of 250 kg/kg, i.v . (a dose producinga more than twofold increase o f the NMDA response in controlrats), failed to produce any potentiation of the NMDA responsein PTX-pretreated rats (Fig. 7A,B). We have reported previouslythat PTX pretreatment abolishes the potentiation of the NMDAresponse by JO-1784, but not that induced by (+)pentazocine,and this ef fec t of (+)pentazocine was still reversed by a low doseof haloperidol (Monnet et al., 1994). In the present series, thepotentiating eff ect of (+)pentazocine (10 pg/kg, i.v. ) was stillpresent in PTX-treated rats; this ef fec t of (+)pentazocine wasreversed readily by progesterone (20 pg/kg, i.v .; Fig. 7C,D).


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0 ControlEl DHEA (250 pglkg, i.v.)

Pregnenolone sulfate(250 pglkg, i.v.)

0 ControlEl Pregnenolone (250 pglk g, Lv.)

DHEA (250 pglkg, i.v.)

Figwe 5. Responsiveness, cxprcsscd as the numhcr of spikesgenerated per nC (mean % SEM), of CA, dorsal hippoc ampu spyramidal neurons to microiontophorctic applications ofq Control l-l Control NMDA before (opejz colunzrz.s) and after (gay coltrr?~~.~) the

El Pregnenolone (250 Kg/ kg, i.v.) El administration of DHEA (A). prcgncnolone (B, C), or DTGDTG (1 Lglkg, Lv.) (D), and after the subsequent administration of prcgncnoloncq DTG (1 pg/kg, iv.) Pregnenolone sulfate (250 pg/k g, i.v.) sulfate (A, D), DHEA (R), or DTG (C) (dmk colwnrzs).Aslerisks indicate p < 0.05 using paired Students / tat.Eff ect of OVX on the potentiation induced by DTGIn the final series of experiments, we measured the magnitudeof the potentiation induced by DTG (I pg/kg, i.v .) in male ratsand in female rats at days 1 and 3 of the menstrual cycle, as wellas 2 weeks after OVX. As shown in Figure 8, no signi ficantdifference was found between the degrees of potentiation in-duced by DTG in male rats and non-OVX female rats at eitherday I or day 3 of the menstrual cycle. In these three groups, thepotentiation of the NMDA response induced by DTG wassuppressed completely by progesterone (20 pg/kg, i.v. ) and byhaloperidol (20 pg/kg, i.v .; Fig. 8). However, the degree of thepotentiation produced by DTG in OVX rats was sign ificant lygreater than that observed in the three other groups (Fig. 8).Moreover, in OVX rats, one dose of 20 pg/kg, i.v ., progester-one reversed DTG-induced potentiation only partially. Such adose of progesterone completely reversed the eff ect of DTG inmale and non-OVX female rats (Fig. 8). In OVX rats, a seconddose of 20 pg/kg, i.v ., progesterone or a subsequent injection of20 pg/kg, i.v. , haloperidol was required to obtain a completesuppression of the potentiation of the NMDA response in-duced by DTG (Fig. 80).

DISCUSSIONThe present results indicate that DHEA at very low doses selec-tively potentiates, in a dose-dependent manner, the neuronalresponse to microiontophoretic applications of NMDA onto py-ramidal neurons in the CA, region of the rat dorsal hippocampus(Fig. 1). This potentiation is reversed b y NE-100 and haloperidol,but not by spiperone or saline (Fig. I). Progesterone, at dosesranging from 1 pg/kg to 2 mgikg, iv. , does not modify the NMDAresponse by itself , but suppresses at the very low dose of 20 pgikg,i.v ., potentiation of the neuronal response to NMDA induced byDHEA as well as by several nonsteroidal (r ligands (Figs. 2-4).Pregnenolone and pregnenolone sulfate neither modify theNMDA response nor prevent or suppress the potcntiation of theNMDA response induced by DHEA and by nonsteroidal (T ago-nists (Fig. 5).

Several interactions between the NMDA-receptor complex andsome neuroactive steroids have been documented (Wu et al.,1991; Irwin et al., 1992; Maione et al., 1992; Bowlby, 1993). Inparticular, pregnenolone sulfate augments NMDA receptor-mediated elevations in intracellular Ca+ in cultured rat hip-pocampal neurons, presumably via a potentiation of the glutama-


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1200 J. Neurosc i., February 1, 1996, 76(3):1193-1202 Bergeron et al. l Neuroactive Steroids and cr Receptors

0 Control 0 ControlEl DHEA (250 pglkg, i.v.) El DTG (20 nA)

DTG (20 nA) DHEA (250 pglkg. i.v.)

F&rr 6. Responsiveness, expressed as the number o f spikes generatedper nC (mean t SEM), of CA, dorsal hippocampus pyramidal neurons tomicroiontophoretic applications of NMDA in control rats before (opencohtmns) and after (~~:I.cI)ol~m~zs) the administrat ion of DHEA (A) orDTG (R), and after the subsequent administrat ion of DTG (A) or DHEA(B) (tlurk col~mrzs). A.stetisks indicate p < 0.05 using paired Students 1test.tergic activat ion of the NMDA receptor (Irwin et al., 1992). In thispreparation, NMDA-receptor activation produces a greater in-ward current of CaZ+ in the presence o f pregnenolone sulfate; thisef fec t has been attributed to a direct action of the steroid on theNMDA-receptor complex (Bowlby, 1993). Pregnenolone sulfatealso enhances NMDA-gated currents in spinal cord neurons andincreases significantly the proconvulsant activ ity of NMDA(Maionc et al., 1992). The mechanisms whereby neurosteroidsaf fec t glutamatergic transmission are not elucidated completely. Agrowing body of evidence suggests that many neuroactive steroidscan rapidly alter the excitabili ty of neurons via a modulation ofGABA, receptors acting as agonists (e.g., pregnenolone sulfateand estrogen) or as antagonists (e.g. , progesterone) (Majewska etal., 1986, 1990; Lambert et al., 1987; Majewska and Schwartz,1987; Mienville and Vicini, 1989; Morrow et al., 1990; Wu et al.,1990; Purdy et al., 1991). The interactions observed in the presentstudy are unlikely to be related to a GABA,-receptor modulation,because pregnenolone and pregnenolone sulfate were inactive inour model (Fig. S), whereas these two neuroactive steroids areamong the most active in paradigms involving the GABA, recep-tors (for review, see Baulicu, 1991). Other mechanisms also havebeen suggested, such as the existence of steroid recognition siteson the NMDA-receptor complex itse lf (Irwin et al., 1992). How-ever, such a mechanism could not account for the ef fec t ofprogesterone in our paradigm, because it did not modify theNMDA response. Nonetheless, the possibility of a downstreamaction of progesterone at the level of effector mechanisms trig-gered by a-receptor activation cannot be ruled out at present.

Several observations suggest that the selective modulation ofthe NMDA response by the neuroactive steroids reported here ismediated via cr receptors. First, the potentiation induced byDHEA is suppressed by haloperidol, but not by spiperone (Fig.1C). Indeed, spiperone binds with high affinit y to dopaminergic,a,-adrenergic, and serotonergic receptors, as does haloperidol(Burt et al., 1977; Clark et al., 1985), but it has a low aff ini ty fora-binding sites (Su, 1982; Tam and Cook, lY84; Weber et al.,1986; Steinfels et al., 1989). Second, a low dose (25 pgikg, i.v .) ofthe selective cr antagonist NE-100 also suppresses the potentiation

q Controlq DHEA (250 pgikg. i.v.)Progesterone (20 pglkg, i.v.)

0 Control

0 PTX pretreatmentq DHEA (250 Kg/kg, i.v.)Progesterone (20 pg/k g, i.v.)

D

201.5

1.0

0.5

0

qq (+)Pentazocine (10 fig/ kg, iv.) pJProgesterone (20 Kg/ kg. i.v.)

PTX- pretreatment(+)Pentazocine (10 wgik g. I.v. )Progesterone (20 pgikg, t.v. )

F@tre 7. Responsiveness, expressed as the number of spikes generatedper nC (mean + SEM), of CA, dorsal hippocampus pyramidal neurons tomicroiontophoretic applications of NMDA in control rats (A, C) and inPTX-treated rats (B, D) before (open cohtmns) and after (~IYI~ col~r~.s)the administration of DHEA (A, B) or (+)pcntazocine (C. U). and afterthe subsequent administration of progesterone (L/O& colunrr~s). A.~twi.sksindicate p < 0.05 using paired Students t test.

of the NMDA response induced by DHEA (Fig. lF), as well as thepotentiation induced by nonsteroidal (T ligands (Debonncl et al.,1995a,b). Third, low doses of progesterone prevent and suppressnot only the potentiating effect of DHEA, but also those inducedby the selective nonsteroidal (T ligands DTG, 50-1784, L-687384,and (+)pentazocine (Figs. 2-4). Fourth, pregnenolone and preg-nenolone sulfate, which have low aff ini ty for (r receptors (Su et al.,1988), neither prevent nor suppress the potentiation of theNMDA response by DHEA and by the nonsteroidal (r agonistseven when administered at doses up to 2 mgikg, i.v . Moreover,our hypothesis is supported by a recent report showing thatneuroactive steroids modulate, via CT eceptors, the [jH]norepi-nephrine release evoked by NMDA in the rat hippocampus (Mon-net et al., 1995).

After the intravenous administration of DHEA (200 pg/kg),microiontophoretic application of DTG (20 nA) was inef fect ive inenhancing the NMDA response further (Fig. 6). Moreover, whenDTG was applied microiontophoretically (20 nA), the intravenousinjection of DHEA was inef fect ive in enhancing the NMDAresponse further (Fig. 6). The occurrence of these bilateral occlu-


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Bergeron et al. . Neuroactive Steroids and u Receotors

cl Control (male)El DTG (1 pg/kg ,i.v.)q Progesterone (20 pglkg, i.v.)

I0 Control (female, day 3)0 DTG (1 pg/kg. I v.)q Progesterone (20 @g/ kg, i.v.)

cl Control (fem ale, day 1)q DTG (1 pg/kg. i.v.)Progesterone (20 pglkg, ix)

0 14 days following OVXEl DTG (1 pg/kg, t.v.)

Progesterone (20 Kg/ kg, i.v.)n Haloperidol (20 Kg/ kg, i.v.)

,?gigl~e 8. Rcsponsivencss, expressed as the number of spikes generatedper nC (mean ? SEM), of CA, dorsal hippocam pus pyramidal neurons tomicroio ntopho rctic app licatio ns of NMDA before (o[~e/z co/rrnzn.s) andafter (gruy colrrmns) the admin istration of DTG and after the subseq uentadmin istration of progesterone (clar-k grrr.v colum~z.s) and halop eridol (blockcolumrz). A.steri.cks indicate p < 0.05 using paired Students t test. tindicates p < 0.0001 comparing the effect of DTG with that in male andnon-OVX fcmalc rats using covariance analysis.

sion phenomena provides additional evidence that the potentia-tion of the NMDA response by DHEA is mediated by cr receptors.

We have reported previously that several nonsteroidal (r ligandsexhibit a bell-shaped dose-response curve (Bergeron et al., 1993,1995a). This e ffe ct does not appear to be attributable to a rapiddesensitization of u receptors but, rather, to the sequential acti-vation of distinct subtypes of (r receptors (Bergeron et al., 199Sa).DHEA does not present such a bell-shaped dose-response curve,but plateaus at doses higher than 500 pg/kg, iv . (Fig. 1B). Thissuggests that DHEA acts on only one subtype of (T receptor. Thefacts that the potcntiating effe ct of DHEA was suppressed by PTXpretreatment and by the selective (7, antagonist NE-100 suggestthat the eff ect of DHEA is mediated via cr, receptors. Moreover,after the inactivation of G,,,-proteins by a PTX pretreatment, theintravenous administration of DHEA fails to modify the neuronalresponse to NMDA (Fig. 6) suggesting that the potentiatingeff ect of DHEA on the NMDA response results from the activa-tion of U, receptors coup led to G,,,,-proteins. We have reported

J. Neurosc i., February 1, 1996, 16(3):1193-1202 1201

previously that PTX pretreatment does not aff ect the potcntiationof the NMDA response induced b y (+)pentazocine, but sup-presses those induced by DTG and JO-1784 (Monnet et al., 1994).Thus, it appears that DHEA activates a different (r, receptor fromthat activated by (+)pentazocine. Interestingly, progesterone re-verses the persistent potentiating ef fec t of (+)pentazocine inPTX-rats (Fig. hD), as is the case for haloperidol (Monnct et al.,1994) which suggests that, in contrast to DHEA, progcstcroncacts on more than one subtype of m receptor. The fac t that thepotentiation of NMDA-induced neuronal response obscrvcd it?vitro by Bowlby (1993) with pregnenolonc sulfate was not sup-pressed after a PTX pretreatment constitutes another argumentsuggesting that pregnenolone sulfate (in his paradigm) andDHEA (in our model) modulate the NMDA response via distinctmechanisms.

No significant difference was found in the degrees of potcntia-tion of the NMDA response induced by DTG in male and non-OVX female rats. However, a significant increase in the magni-tude of this potentiation was observed in OVX rats (Fig. 7). Thismay be attributable, at least in part, to the fac t that OVX de-creases the levels of progesterone. Indeed, because this steroidappears to be a potent antagonist of (r receptors, the low levels ofprogesterone in the OVX rats may account for the greater poten-tiation of the NMDA response by DTG. This would imply that theNMDA response is dampened tonically by progesterone. In keep-ing with this interpretation, there is a marked reduction in theeffectiveness of DTG in potentiating the NMDA response duringlate pregnancy (Bergeron et al., 1995b), a period during whichprogesterone levels are ver y high (Schwarz et al., 1989). Given thevery low doses of progesterone administered in the present study,one can assume that its eff ect is of physiological relevance.

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richard bergeron, claude de montigny and guy debonnel- potentiation of neuronal nmda response...

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