Potentiation of Neuronal NMDA Response Induced by Dehydroepiandrosterone and Its Suppression by Progesterone: Effects Mediated Via Sigma Receptors

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

Potentiation of Neuronal NMDA Response Induced by Dehydroepiandrosterone and its Suppression by Progesterone: Effects Mediated via Sigma Receptors

Richard Bergeron, Claude de Montigny, and Guy Debonnel Department of Psychiatry, Neurobiological Psychiatry Unit, McGill University, Montreal, Quebec, Canada H3A 1A 1

We have shown previously that low doses of selective sigma well as those induced by nonsteroidal u ligands. Neither preg- (cr)-receptor ligands potentiate the excitatory response of py- nenolone nor pregnenolone sulfate had any effect on the NMDA ramidal neurons to NMDA in the CA, region of the dorsal response-nor did they antagonize the potentiation of the hippocampus in the rat. Because progesterone competitively NMDA response induced by DHEA and by nonsteroidal IJ displaces the binding of the ligand N-[3H]allyl-normetazocine ligands. A pet-tussis toxin pretreatment, which inactivates G,,,- (SKF-10,047), the present studies were undertaken to deter- proteins, abolished the potentiating effects of DHEA. Ovariec- mine in viva the effect of neuroactive steroids on NMDA- tomy enhanced the potentiation of the NMDA response by the induced excitation of rat CA, pyramidal neurons. Low doses of nonsteroidal CT ligand di(2-tolyl)guanidine (DTG). There was a dehydroepiandrosterone (DHEA) potentiated the NMDA re- reciprocal occlusion of the effects of DHEA and DTG; DTG did sponse selectively and dose-dependently. The effect of DHEA not potentiate the NMDA response further after DHEA, and was reversed by the selective v antagonist N-dipropyl-2-(4- DHEA did not do so after DTG. These results suggest that some methoxy-3-(2-phenylethoxy)phenyl)-ethylamine monohydro- neuroactive steroids modulate the NMDA response via g chloride (NE-100) and by haloperidol, but not by spiperone. receptors. Progesterone had no effect by itself but reversed, at low doses, Key words: neurosteroids; hippocampus; electrophysiology; the potentiation of the NMDA response induced by DHEA as ovariectomy; haloperidol; DTG

Sigma ((7) receptors are present in high density in the CNS Dekleva, 1987), but not by spiperone, which has a binding profile similar (Walker et al., 1990) and in peripheral organs, with a particularly to that of haloperidol except for its low affinity for v receptors (Monnet high density in the ovaries (Wolfe et al., 1989). Many psychotropic et al., 1990, 19Y2). We also have tested recently the novel and very drugs such as antipsychotics and antidepressants have high affinity selective (T ligand N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl)- for v receptors (Su, 1982; Schmidt et al., 1989; Snyder and ethylamine monohydrochloride (NE-100) (Okuyama et al., 1993). It Largent, 1%‘); ltzhak and Kassim, 1990; Ferris et al., 1991). proved extremely potent in blocking or reversing the potentiation of the Several steroids, progesterone in particular, also bind with high NMDA response induced by v ligands such as DTG (Debonnel et al., affinity to (7 receptors (Su et al., 1988). 1995a). Therefore, g ligands acting like DTG have been denoted ten- Previous studies have demonstrated that low doses of selective CT tatively cr “agonists,” and (r ligands acting like haloperidol have been ligands, such as di(2-tolyl)guanidine (DTG) (Weber et al., 1986), denoted “antagonists” (Monnet et al., 1990). These data and several I-benzylspiro[ 1,2,3,4-tetrahydro-aphthalene-1,4-piperidine] (L-687,384) other reports from other laboratories using biochemical, neuroendocri- (Middlemiss ct al., 1991; Barnes et al., 1992), (+)N-cyclopropylmethyl- nological, and behavioral models suggest the existence of a functional N-methyl-l ,4-diphenyl-1-ethyl-butyl-3-N-I-ylamine hydrochloride (JO- interaction between (T and NMDA receptors, although the exact mech- 1784) (Roman et al., 19Y0), and (+)pentazocine (Steinfels et al., 1988) anism is not understood fully (for review, see Dcbonncl, lYY3). selectively potentiate the response of rat CA, dorsal hippocampus py- There are several types of (T receptors; those classified as (r, and ramidal neurons to microiontophoretic applications of NMDA (Monnet cr, have been characterized most extensively (Quirion et al., 1992). et al., 1990, 1992; Martin et al., 1992; Walker and Hunter, 1994). This The most commonly used g ligands, including haloperidol and effect is reversed by other u ligands such as haloperidol, (+)3- DTG, do not discriminate between CT,and (r7 receptors (Quirion hydroxyphenyl-N-i-( 1-propyl)piperidine [also known as (+)3-PPP], et al., 1992), whereas drugs such as (+)pentazocine, JO-1784, and cr-(4-fluorc~phenyl)-4-(5-flu~~ro-2-pyrimidinyl)-l-piper~ine butanol L-687,384, N-allyl-normetazocine [( +)SKF-100471, and NE-100 (BMY-14802) (Largent et al., 1984; Tam and Cook, 1984; Taylor and are selective for g, receptors (Quirion et al., 1992; Chaki et al., 1994). However, recent data obtained in our laboratory have shown that pertussis toxin (PTX) pretreatment abolishes the Rrceivcd Aug. 8, lYY5; rcviscd Oct. IO, IYYS: accepted Nov. I I, 199.5. This work ws supported by the Medical Rcscarch Council of Canada, the Royal potentiation induced by JO-1784, but not that induced by (+)pen- Victoria Hospital Research Institute, and the Fends de la Recherche en SantC du tazocine (Monnet et al., 1994), which suggests that these two (r Ou&ec (FRSO). G.D. received II scholarshiD from the FRSQ, and R.B. received a ligands act on different u-receptor subtypes. Moreover, the injec- fellowship from the FRSO. We thank T.‘Vo for computer programming and statistical ;malyses. C. Bouchard for illustrations, and L. Martin for secrctxial tion of colchicine in the dentate gyrus, which destroys the mossy a\\i\tancc. fiber system (a major afference to CA, pyramidal neurons), abol- Corre\pondcnce should he addressed to Richard Bergcron, Department of Psy- chiatry. Neurohmlogical Psychiatry Umt, McGill UnivcrGty, IO.13 Pint Avenue West. ishes the potentiation of the NMDA response induced by DTG Montreal. Quchec, C;mada H3A I Al. and JO-1784, but not that of (+)pentazocine, suggesting that the Copyright 6: IYYO Society for Ncuroscicnce 0270-6474/9(,/lhl lY3-lO$OS.OO/O subtype of u receptors on which DTG and JO-1784 are acting is 1194 J. Neurosci., February 1. 1996, 76(3):1193-1202 Bergeron et al. l Neuroactive Steroids and o Receptors located prcsynaptically on the mossy-fiber terminal, whereas the maintained at 37°C throughout the experiment. After removal of the dura subtype of u receptors on which (+)pentazocine is acting is mater, the micropipcttc was lowered into the CA, region of the dorsal hippocampus (lateral, +4.2 mm; anterior, +4.2 mm from lambda: dorsal. located postsynaptically (Debonnel et al., 199%). -3.5 to -4.5 mm from the cortical surface). Pyramidal neurons wcrc Several reports have shown that progesterone acts as a competitive identified by their long-duration (11.X-1.5 mscc) and large-amplitude (0.5-2 inhibitor of [3H](+)SKF-10047 or [‘H]haloperidol (Su et al., 1988; mV) action potentials and by the presence of characteristic complex spike iLk&nn et al., 19Y4; Yamada et al., 1994; Ramamoorthy et al., 1995) discharges alternating with simple spike activity (Kandcl and Spencer. lOhI). at (r receptors and have prompted us to assess the potential agonistic Neuronal firing activity was monitored via an oscilloscope after signal magnification by a high-input impedance amplifier. Action potentials were or antagonistic properties of progesterone, dehydroepiandrosterone detected by a differential amplitude discriminator generating square pulses, (DHEA), and other neuroactive steroids (Paul and Purdy, 1992) in which were stored on-line and forwarded to a paper chart rccordcr gcner- an bz r,it~ clectrophysiological paradigm. sting integrated firing-rate histograms. Alternatc microiontophorctic applications of SO set of each excitatory substance (NMDA. QUIS, and ACh) MATERIALS AND METHODS separated by SO set retention periods wcrc carried out continuously during The experiments were carried out i/z t,il~ in the CA, region of the rat the duration of the recording. The duration of the microiontophorctic dorsal hippocampus, in which the responsiveness of pyramidal neurons to applications and the intensity of the currents used also wcrc stored on-line. microiontophorctic applications of NMDA, quisqualate (QLJIS), and The elfccts of their applications on pyramidal neuron firing activity were acetylcholine (ACh) was assessed using extraccllular unitary recordings. expressed as the number of spikes generated per nanocoulomb (nC: I nC is P~e~~amtiorr qf rr/~ir?ials. Male Sprague-Dawlcy rats weighing 200-250 the charge generated by 1 nA applied for 1 set). gm were ohtaincd from Charles River Laboratories (St. Constant, Que- After a neuron was isolated, it was recorded for a period of at least 20 bec. Canada). Female rats of the same weight wcrc obtained at day 1 or min before any drug was injected. The recording was stored on-line .? of the menstrual cycle or 2 weeks after ovariectomy (OVX). Rats were without interruption for the duration of the experiment. Five to six housed three to four per cage with free access to food and water. They applications of each excitatory substance (NMDA. QUIS. and ACh) wcrc were maintained at constant temperature (25°C) under a I2 hril2 hr carried out before the drugs studied wcrc injcctcd or applied microion- light/dark cycle. tophoretically. The effects of drugs studied appcarcd within a period of 10 PTX~“‘t~ecrt,,le,ft. The pretreatment with PTX (I pg in 2 /.LI of physi- min after their intravenous injection. The calculations wcrc carried out ological saline; Sigma, St. Louis, MO) consisted of lowering the tip of a when the maximal effect of the drug was achieved (within the first 20 5 ~1 Hamilton syringe unilaterally into the right dorsal hippocampus of min). At the end of the experiment, a -27 FA current was passed through chloral hydrate-ancsthctizcd rats (anterior, 4.5 mm; lateral, 4 mm; and the central barrel for 20 min to deposit Fast Green FCF for subscqucnt dorsal, 4 mm) according to the atlas of Paxinos and Watson (1986). PTX histological verification of recording sites. was injcctcd slowly over a period of5 min. Control rats were injected with Esl,crinrerzral se+s. Neuroactive steroids wcrc prcparcd in ;I solution of an equal volume ofphy\iological saline under the same conditions. In i~ir~o 40% polyethylene glycol. The (r ligands were prepared in physiological clectrophysiological cxperimcnts were carried out S-7 d later. saline, and they were administered via a lateral tail vein. In all cxpcri- Drir~,s, The following substances were used: DTG (Aldrich, Milwaukee, mental series, only one dose of each drug was administered to one rat WI); 50-1784, a gift from J. Junien (Institut de Rechcrche Jouvcinal, while recording from one neuron. Fresncs, France); L-687,384, a gift from L. Iversen (Merck-Sharp and In the first series of experiments, several doses of each drug wcrc tested to Dohmc, Tyler Park, UK); NMDA and ACh (Sigma): QUIS (Tocris generate dose-response ctnvcs. Progesterone, DHEA, prcgnenolonc, and Neuramin, Buckhurst Hill, Essex, UK): haloperidol (McNeil Lahorato- pregnenolone sulfate wcrc tested at doses ranging from I Kg to 2 mgikg. In ries, Stoulfville, Ontario, Canada): spiperonc, (+)pcntazocine, progcster- the second series, progesterone (20 pg/kg, iv.) was tested as an antagonist of one. prcgnenolonc, prcgnenolone-sulfate, and DHEA (Research Bio- the potcntiation of the NMDA response induced by the previous adminis- chemicals, Natick. MA); NE-100, a gift from S. Okuyama (Taisho tration of a low dose of DHEA or of the following nonstcroid rr ligands: Pharmaceutical. Ohmiya, Japan). DTG I pg/kg, i.v.; L-(,X7,384 I kg/kg, iv.; JO-17X4 5 &kg, iv.; (+)penta- Prc~,clrtr/iorr of’ irric.r.o~>i/~e/lc.s. Microiontophorctic applications and cx- zocine IO &kg, iv. In the third series, rats were pretrcatcd with 21 local tracellular unitary recordings were performed with S-barrel glass micropi- injection of PTX to assess the possible involvement of (r rcccptors coupled pettcs prepared in a conventional manner (Haigler and Aghajanian, with G,,,,-proteins in the potentiation of the neuronat response to NMDA bv 1974). Three of the side barrels used for microiontophorcsis contained neuroactive steroids. In the fourth series, we have examined the possibility 01 (in mM): NMDA IO (in NaCI 20(l), pH 8, QUIS I.5 (in NaCl 400), pH X, a reciprocal occlusion of the cffccts of DTG and DIIEA. In the final scrics. and ACh 20 (in NaCI 200), pH 4. The remaining side barrel, used for the degree of the potcntiation of the NMDA response induced by DTG (I automatic current balancing, and the central barrel, used for extracellular &kg, iv.) was measured in male rats, in female rats at days I and .i of the unitary recording, were lillcd with 2 M NaCI saturated with Fast Green menstrual cycle, and in OVX rats I4 d after the surgery. (FCF; Aldrich). Crrk~irkrtions. The computer calculated the clfect of each 50 SK micro- Rwordiq.t J?ot11 CA., rlor:wl hip,uocanyx~s pynn~iclul NCLIK~IIS. For the iontophoretic application of an excitatory substance as the total numbct electrophysiological experiments, rats were ancsthetizcd with urethane ( I.25 of spikes generatcd/nC. Each value was calculated by the computer as the g/kg, ip.) and mounted in a stcreotaxic apparatus. Body tcmpcrature was mean of the effect of three consecutive 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 applications of NMDA and QUlS before and after the injection of DHEA (crr~ow al /cft) and after the subsequent administration of haloperidol (~~~oM.(I/ rig/i/). In this and the subsequent integrated tiring-rate histograms, bars indicate the duration of applications for which currents arc given (in nA), and o,>c,i circks reprcscnt an interruption of the illustration of the continuous recording. R, Dose-response curve of the cl&t of the intravenous administration of DHEA on the neuronal activation of CA, dorsal hippocampus pyramidal neurons induced by microiontophoretic applications of NMDA. Each c/or rcprcscnts the effect of one dose of the drug administered to one rat while recording from one neuron in this and subsequent doseresponse curves. The clfcct was assessed by determining the ratio (N,:N,) of the number of spikes gcneratcd per nC of NMDA before (N,) and after (NJ) the injection of the drug. C, Responsiveness. cxprcssed as the number of spikes generated per nC (mean 2 SEM), of CA, dorsal hippocampus neurons to microiontophorctic applications of NMDA before (o/jen cohmms) and after (grcry cohrm/r.s) the administration of DHEA and after the subscqucnt administration of halopcridol (&I& cohmzr~.s). The rrrr&er- within the ope,r colrrnirzs indicates the number of neurons tcstcd (I neuron/rat in this and subscqucnt bar chart histograms). D, Rcsponsivencss, expressed as the number of spikes generated per nC (mean t SEM), of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (opefz cohrmns) and after (fi’Ny co/rrrn,zs) the administration of DHEA and after the subscqucnt administration of spiperonc ((lurk cohrnm~). The nwnher- within the o,lerl colltr~zn indicates the number of neurons tested (1 neuron/rat in this and subsequent bar chart histograms). E, Responsiveness, expressed as the number of spikes gcncrated per nC (mean ? SEM), of CA, dorsal hippocampus neurons to microiontophorctic applications of NMDA before (oven colrtrnns) and after @uy cohrm!z.s) the administration of DHEA and after the subsequent administration of saline (&irk col7m7r7.s). F, Responsiveness, expressed as the number of spikes generated per nC (mean i- SEM). of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (o/le,i co/rrm,z.s) and after (grrry colnnnr,s) the administration of DHEA and after the subsequent administration of the selcctivc (7 antagonist NE-100 (t/t?& coh~~n~i.s). A.s~ri,sks in C-F indicate 1’ < 0.05 using paired Student‘s I test. Bergeron et al. . Neuroactive Steroids and CT Receptors J. Neurosci., February 1, 1996, 16(3):1193-1202 1195

A B QUIS NMDA 3- . . -5 -12 200 mu uzauE7zac7 m z 2- -a 1 I, . . *. i I-J- Y a WI 0 00 00 OJ I 'u-l 100 1 0.1 1 10 100 1000 10000 t t 1 min Dose (Kg/kg, I.v.) of DHEA DHEA Haloperidol 100 pg/kg, i.v. 10 @g/kg, i.v.

x D 2.0

1.5

1.0

0.5

cl Control III Control DHEA (250 pg/kg, Lv.) DHEA (250 pglkg, i.v.)

Haloperidol (20 pg/kg, i.v.) Spiperone (20 pg/kg, i.v.)

F * x 2.0 x

1.5

1 .o

0.5

cl Control 0 Control DHEA (250 pg/kg, i.v.) DHEA (250 pg/kg, i.v.)

Salin I NE-100 (25 pglkg, i.v.) 1196 J. Neurosci., February 1, 1996, 16(3):1193-1202 Bergeron et al. . Neuroactive Steroids and CT Receptors

A ACh QUIS NMDA 9 -5 -11 I-0 I-0 IDO

; 100 x E-7

‘1 0 00

+ -2 min Progesterone Halopendol 100 pg/kg, i.v. 20 Kg/kg, i.v.

* . . . 1 . . . . .

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

2.0

1.5 Figwe 2. A, Integrated firing-rate histogram of a CA, dorsal hippocampus pyramidal neuron illustrating the effect of microiontophoretic applications of NMDA, QU/S, 1.0 and AC/? before and after the injection of progesterone (~770~ (71 I@) and after the subsequent injection of haloperidol (nrrow ut ri& see legend to Fig. 1). B, Dose- 0.5 response curve of the effect of the injection of progesterone on the neuronal activation of CA, dorsal 0 hippocampus pyramidal neurons induced by microiontophoretic applications of NMDA. C, Responsiveness, expressed as the number of spikes generated per nC (mean + SEM), of CA, dorsal hippocampus pyramidal neurons to microiontophoretic applications of NMDA before Control cl Control (open columns) and after (gray cobrmns) the administra- DHEA (250 pg/kg, i.v.) Progesterone (20 pg/kg, i.v.) tion of DHEA or progesterone and after the subsequent administration of either steroid (dark columns). Asterisk Progesterone (20 pg/kg, i.v.) DHEA (250 pglkg, i.v.) indicates p < 0.05 using paired Student’s t test. substances. The effects of the intravenous administration of the drugs significance was assessed using Student’s t test with Dunnett’s correction studied were assessed when the maximal effect was achieved, by deter- for multiple comparisons. Covariance analysis was used to compare the mining the ratio (N?:N,) of the number of spikes generated/nC of each of degree of the potentiation of the NMDA response induced by DTG (1 the three excitatory substances ACh, QUIS, and NMDA before (N,) and Kg/kg, i.v.) in male and in non-OVX and OVX female rats; p 5 0.05 was after (N,) the injection of the drug. The dose-response curves of the considered significant. effects of intravenous administration of CT ligands were obtained by fitting experimental data to general logistic equations obtained with the soft- RESULTS ware Tablecurve (version 3.0, Jandel Scientific, San Rafael, CA). Sruristical ~7u7IJI~e.s. All results are expressed as mean % SEM of the All recordings were obtained from the stratum pyramidale of the number of spikes gencrated/nC of NMDA, QUIS, or ACh. Statistical dorsal hippocampal CA, region, as confirmed by histological Bergeron et al. . Neuroactive Steroids and (r Receptors J. Neurosci., February 1, 1996, 76(3):1193-1202 1197

ACh QUIS NMDA 7 -2 -12 ILzi2oImao~mao-rrrao-rzzao

200 -

03 0 3j y loo- a cn

DTG 1 pglkg, i Y ACh QUIS NMDA 7 -2 -12 IilL;loIQzao~!zaomrrrao~arao

200 -

LA 9 2 2 loo- EL m

Progesterone 20 pg/kg, i Y ACh QUIS NMDA

Figure 3. Continuous integrated tiring- rate histogram of a CA, dorsal hippocampus pyramidal neuron illustrating the effect of microiontophoretic applications of NMDA, QUIS, and AC% before and after the administration of DTG (arrows) and after the administration of progesterone. Time scale applies to the 1 min three traces.

verification of the Fast Green FCF deposit left at the end of each potentiation was obtained by increasing the dose of DHEA up to experiment. 2 mg/kg, i.v. (Fig. 1). This enhancing effect of DHEA lasted for at The intravenous injection of the vehicle (40% polyethylene least 40 min. The potentiation of the NMDA response by DHEA glycol), used for preparing the solutions of neuroactive steroids, was reversed by haloperidol (20 pg/kg, i.v.; Fig. lC), but not by affected neither the spontaneous firing activity of dorsal hip- spiperone (20 kg/kg, i.v.; Fig. 1D) and not by the intravenous pocampus CA, pyramidal neurons nor their response to NMDA, administration of saline (Fig. 1E). Moreover, NE-100, a novel QUIS, and ACh. selective v antagonist (Okuyama et al., 1993) at a dose of 25 pg/kg, i.v., also suppressed the potentiation of the NMDA re- Effects of DHEA sponse induced by DHEA (Fig. 1F). The intravenous administration of 100 pg/kg DHEA induced a twofold increase of the response of dorsal hippocampal CA3 Effects of progesterone pyramidal neurons to microiontophoretic applications of NMDA At doses ranging from 1 to 2000 pg/kg, i.v., progesterone did not without affecting their response to QUIS (Fig. 1A). The effect of affect the neuronal response induced by microiontophoretic ap- DHEA was dose-dependent, and a maximal potentiation was plications of NMDA (Fig. 2&?), QUIS, or ACh. However, pro- obtained at a dose of 500 pg/kg, i.v. (Fig. 1B). No additional gesterone, at a dose of 20 kg/kg, i.v., prevented and reversed the 1198 J. Neurosci., February 1, 1996, 76(3):1193-1202 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 effect of progesterone could be mediated via c receptors, low doses of the selective 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 Figure 3. Progesterone (20 pg/kg, i.v.; Fig. 4A-D) reversed the potentiation induced by the nonsteroidal v ligands. Haloperidol (20 pg/kg, i.v.; Fig. 4E), but not spiperone (20 pg/kg, i.v.; Fig. 4F), also reversed the effect of DTG as reported previously (Monnet et al., 1990; Bergeron et al., 1993). Effects of pregnenolone and pregnenolone sulfate 0 CUltKi cl Control The effects of pregnenolone and pregnenolone sulfate were as- DTG (1 fig/kg. i.v.) JO-1784 (5 pglkg, i.v.) sessed in the same paradigm. At doses ranging from 1 to 2000 w Progesterone (20 Kg/kg, iv.) Progesterone (20 pglkg, i.v.) pg/kg, i.v., these two neuroactive steroids did not modify NMDA-, QUIS-, or ACh-induced neuronal responses. The efficacy of these two neuroactive steroids in reversing the potentiation of the NMDA response induced by a previous administration of DHEA D (100 Fg/kg, i.v.), as well as by other nonsteroidal (T agonists, also 0 was tested. Pregnenolone and pregnenolone sulfate neither re- 2.0 versed nor prevented the potentiation of the NMDA response induced by DHEA (Fig. 5A,B). Similarly, neither pregnenolone 1.5 nor pregnenolone sulfate suppressed the potentiation of the NMDA response induced by the nonsteroidal cr agonists DTG (1 1.0 Fg/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 the 0.5 latter three compounds). 0 Effect of coadministration of DHEA and DTG To test the possibility that DHEA and nonsteroidal rr ligands both Cl Control 0 Control activate (T receptors, we have examined the possibility of a reciprocal occlusion of the effects of DHEA and DTG. The microion- q (+)Pentazocine (10 pg/kg, i.v.) /#f L-687,384 (1 pglkg, i.v.) tophoretic applications of DTG (20 nA) produced, as observed n Progesterone (20 Kg/kg, i.v.) H Progesterone (20 Kg/kg, i.v.) previously (Monnet et al., 1990; Bergeron et al., 1995a), a twofold increase in the neuronal response to NMDA. The injection of DHEA at 200 pg/kg, i.v., failed to elicit an increase in NMDA response. Conversely, the intravenous administration of DHEA at 200 pg/kg also induced a threefold increase in the neuronal * * response to NMDA, and the subsequent microiontophoretic applications of DTG (20 nA) failed to elicit an increase in NMDA response (Fig. 6). Effect of PTX pretreatment on the potentiation induced by DHEA PTX, which inactivates G,,-proteins via ADP ribosylation, was used to assess the possible involvement of these proteins in the modulation of NMDA-induced neuronal activation in the CA, region of the rat dorsal hippocampus by DHEA. The in viva PTX pretreatment affected neither the spontaneous firing activity of 0 Control Cl Control CA, pyramidal neurons nor their responsiveness to NMDA, fg DTG (1 pglkg, i.v.) q DTG (1 fig/kg, i.v.) QUIS, or ACh, which is in agreement with previous data (Monnet et al., 1994). DHEA, at a dose of 250 kg/kg, i.v. (a dose producing I Haloperidol (20 kg/kg, iv.) Spiperone (20 Kg/kg, i.v.) a more than twofold increase of the NMDA response in control

Figure 4. Responsiveness, expressed as the number of spikes generated rats), failed to produce any potentiation of the NMDA response per nC (mean ? SEM), of CA, dorsal hippocampus pyramidal neurons to in PTX-pretreated rats (Fig. 7A,B). We have reported previously microiontophoretic applications of NMDA before (open columns) and that PTX pretreatment abolishes the potentiation of the NMDA after (gray columns) the intravenous administration of DTG (A). JO-1784 response by JO-1784, but not that induced by (+)pentazocine, (B), (+)pentazocine (C), and L-687384 (D), and after the subsequent intravenous administration of progesterone (A-D), haloperidol (E), or and this effect of (+)pentazocine was still reversed by a low dose spiperone (F) (dark columns). Asterisks indicate p < 0.05 using paired of haloperidol (Monnet et al., 1994). In the present series, the Student’s t test. potentiating effect of (+)pentazocine (10 pg/kg, i.v.) was still present in PTX-treated rats; this effect of (+)pentazocine was reversed readily by progesterone (20 pg/kg, i.v.; Fig. 7C,D). Eiergeron et al. . Neuroactive Steroids and (r Receptors J. Neurosci., February 1, 1996, 16(3):1193-1202 1199

0 Control 0 Control El DHEA (250 pglkg, i.v.) El Pregnenolone (250 pglkg, Lv.)

Pregnenolone sulfate DHEA (250 pglkg, i.v.) (250 pglkg, i.v.)

Figwe 5. Responsiveness, cxprcsscd as the numhcr of spikes generated per nC (mean % SEM), of CA, dorsal hippocampus pyramidal neurons to microiontophorctic applications of q Control l-l Control NMDA before (opejz colunzrz.s) and after (gay coltrr?~~.~) the administration of DHEA (A). prcgncnolone (B, C), or DTG El Pregnenolone (250 Kg/kg, i.v.) El DTG (1 Lglkg, Lv.) (D), and after the subsequent administration of prcgncnolonc sulfate (A, D), DHEA (R), or DTG (C) (dmk colwnrzs). q DTG (1 pg/kg, iv.) Pregnenolone sulfate (250 pg/kg, i.v.) Aslerisks indicate p < 0.05 using paired Student’s / tat.

Effect of OVX on the potentiation induced by DTG DISCUSSION In the final series of experiments, we measured the magnitude The present results indicate that DHEA at very low doses selec- of the potentiation induced by DTG (I pg/kg, i.v.) in male rats tively potentiates, in a dose-dependent manner, the neuronal and in female rats at days 1 and 3 of the menstrual cycle, as well response to microiontophoretic applications of NMDA onto py- as 2 weeks after OVX. As shown in Figure 8, no significant ramidal neurons in the CA, region of the rat dorsal hippocampus difference was found between the degrees of potentiation in- (Fig. 1). This potentiation is reversed by NE-100 and haloperidol, duced by DTG in male rats and non-OVX female rats at either but not by spiperone or saline (Fig. I). Progesterone, at doses day I or day 3 of the menstrual cycle. In these three groups, the ranging from 1 pg/kg to 2 mgikg, iv., does not modify the NMDA potentiation of the NMDA response induced by DTG was response by itself, but suppresses at the very low dose of 20 pgikg, suppressed completely by progesterone (20 pg/kg, i.v.) and by i.v., potentiation of the neuronal response to NMDA induced by haloperidol (20 pg/kg, i.v.; Fig. 8). However, the degree of the DHEA as well as by several nonsteroidal (r ligands (Figs. 2-4). potentiation produced by DTG in OVX rats was significantly Pregnenolone and pregnenolone sulfate neither modify the greater than that observed in the three other groups (Fig. 8). NMDA response nor prevent or suppress the potcntiation of the Moreover, in OVX rats, one dose of 20 pg/kg, i.v., progester- NMDA response induced by DHEA and by nonsteroidal (T ago- one reversed DTG-induced potentiation only partially. Such a nists (Fig. 5). dose of progesterone completely reversed the effect of DTG in Several interactions between the NMDA-receptor complex and male and non-OVX female rats (Fig. 8). In OVX rats, a second some neuroactive steroids have been documented (Wu et al., dose of 20 pg/kg, i.v., progesterone or a subsequent injection of 1991; Irwin et al., 1992; Maione et al., 1992; Bowlby, 1993). In 20 pg/kg, i.v., haloperidol was required to obtain a complete particular, pregnenolone sulfate augments NMDA receptor- suppression of the potentiation of the NMDA response in- mediated elevations in intracellular Ca’+ in cultured rat hip- duced by DTG (Fig. 80). pocampal neurons, presumably via a potentiation of the glutama- 1200 J. Neurosci., February 1, 1996, 76(3):1193-1202 Bergeron et al. l Neuroactive Steroids and cr Receptors

q Control 0 PTX pretreatment Control 0 Control 0 q DHEA (250 pgikg. i.v.) q DHEA (250 Kg/kg, i.v.) El DHEA (250 pglkg, i.v.) El DTG (20 nA) Progesterone (20 pglkg, i.v.) Progesterone (20 pg/kg, i.v.) DTG (20 nA) DHEA (250 pglkg. i.v.)

F&rr 6. Responsiveness, expressed as the number of spikes generated per nC (mean t SEM), of CA, dorsal hippocampus pyramidal neurons to microiontophoretic applications of NMDA in control rats before (open D cohtmns) and after (~~:I.cI)col~m~zs) the administration of DHEA (A) or DTG (R), and after the subsequent administration of DTG (A) or DHEA 20 (B) (tlurk col~mrzs). A.stetisks indicate p < 0.05 using paired Student’s 1 test. 1.5 tergic activation of the NMDA receptor (Irwin et al., 1992). In this 1.0 preparation, NMDA-receptor activation produces a greater in- ward current of CaZ+ in the presence of pregnenolone sulfate; this 0.5 effect has been attributed to a direct action of the steroid on the 0 NMDA-receptor complex (Bowlby, 1993). Pregnenolone sulfate also enhances NMDA-gated currents in spinal cord neurons and increases significantly the proconvulsant activity of NMDA 0 Control q PTX- pretreatment pJ (Maionc et al., 1992). The mechanisms whereby neurosteroids q (+)Pentazocine (10 fig/kg, iv.) (+)Pentazocine (10 wgikg. I.v.) affect glutamatergic transmission are not elucidated completely. A Progesterone (20 Kg/kg. i.v.) Progesterone (20 pgikg, t.v.) growing body of evidence suggests that many neuroactive steroids can rapidly alter the excitability of neurons via a modulation of F@tre 7. Responsiveness, expressed as the number of spikes generated GABA, receptors acting as agonists (e.g., pregnenolone sulfate per nC (mean + SEM), of CA, dorsal hippocampus pyramidal neurons to and estrogen) or as antagonists (e.g., progesterone) (Majewska et microiontophoretic applications of NMDA in control rats (A, C) and in al., 1986, 1990; Lambert et al., 1987; Majewska and Schwartz, PTX-treated rats (B, D) before (open cohtmns) and after (~IYI~ col~r~.s) the administration of DHEA (A, B) or (+)pcntazocine (C. U). and after 1987; Mienville and Vicini, 1989; Morrow et al., 1990; Wu et al., the subsequent administration of progesterone (L/O& colunrr~s). A.~twi.sks 1990; Purdy et al., 1991). The interactions observed in the present indicate p < 0.05 using paired Student’s t test. study are unlikely to be related to a GABA,-receptor modulation, because pregnenolone and pregnenolone sulfate were inactive in our model (Fig. S), whereas these two neuroactive steroids are of the NMDA response induced by DHEA (Fig. lF), as well as the among the most active in paradigms involving the GABA, recep- potentiation induced by nonsteroidal (T ligands (Debonncl et al., tors (for review, see Baulicu, 1991). Other mechanisms also have 1995a,b). Third, low doses of progesterone prevent and suppress been suggested, such as the existence of steroid recognition sites not only the potentiating effect of DHEA, but also those induced on the NMDA-receptor complex itself (Irwin et al., 1992). How- by the selective nonsteroidal (T ligands DTG, 50-1784, L-687384, ever, such a mechanism could not account for the effect of and (+)pentazocine (Figs. 2-4). Fourth, pregnenolone and preg- progesterone in our paradigm, because it did not modify the nenolone sulfate, which have low affinity for (r receptors (Su et al., NMDA response. Nonetheless, the possibility of a downstream 1988), neither prevent nor suppress the potentiation of the action of progesterone at the level of effector mechanisms trig- NMDA response by DHEA and by the nonsteroidal (r agonists gered by a-receptor activation cannot be ruled out at present. even when administered at doses up to 2 mgikg, i.v. Moreover, Several observations suggest that the selective modulation of our hypothesis is supported by a recent report showing that the NMDA response by the neuroactive steroids reported here is neuroactive steroids modulate, via CTreceptors, the [jH]norepi- mediated via cr receptors. First, the potentiation induced by nephrine release evoked by NMDA in the rat hippocampus (Mon- DHEA is suppressed by haloperidol, but not by spiperone (Fig. net et al., 1995). 1C). Indeed, spiperone binds with high affinity to dopaminergic, After the intravenous administration of DHEA (200 pg/kg), a,-adrenergic, and serotonergic receptors, as does haloperidol microiontophoretic application of DTG (20 nA) was ineffective in (Burt et al., 1977; Clark et al., 1985), but it has a low affinity for enhancing the NMDA response further (Fig. 6). Moreover, when a-binding sites (Su, 1982; Tam and Cook, lY84; Weber et al., DTG was applied microiontophoretically (20 nA), the intravenous 1986; Steinfels et al., 1989). Second, a low dose (25 pgikg, i.v.) of injection of DHEA was ineffective in enhancing the NMDA the selective cr antagonist NE-100 also suppresses the potentiation response further (Fig. 6). The occurrence of these bilateral occlu- Bergeron et al. . Neuroactive Steroids and u Receotors J. Neurosci., February 1, 1996, 16(3):1193-1202 1201

previously that PTX pretreatment does not affect the potcntiation of the NMDA response induced by (+)pentazocine, but suppresses those induced by DTG and JO-1784 (Monnet et al., 1994). Thus, it appears that DHEA activates a different (r, receptor from that activated by (+)pentazocine. Interestingly, progesterone re- verses the persistent potentiating effect of (+)pentazocine in PTX-rats (Fig. hD), as is the case for haloperidol (Monnct et al., 1994) which suggests that, in contrast to DHEA, progcstcronc acts on more than one subtype of m receptor. The fact that the potentiation of NMDA-induced neuronal response obscrvcd it? vitro by Bowlby (1993) with pregnenolonc sulfate was not suppressed after a PTX pretreatment constitutes another argument cl Control (male) cl Control (female, day 1) suggesting that pregnenolone sulfate (in his paradigm) and q El DTG (1 pg/kg ,i.v.) DTG (1 pg/kg. i.v.) DHEA (in our model) modulate the NMDA response via distinct mechanisms. q Progesterone (20 pglkg, i.v.) Progesterone (20 pglkg, ix) No significant difference was found in the degrees of potcntiation of the NMDA response induced by DTG in male and non- OVX female rats. However, a significant increase in the magnitude of this potentiation was observed in OVX rats (Fig. 7). This may be attributable, at least in part, to the fact that OVX de- creases the levels of progesterone. Indeed, because this steroid appears to be a potent antagonist of (r receptors, the low levels of progesterone in the OVX rats may account for the greater potentiation of the NMDA response by DTG. This would imply that the NMDA response is dampened tonically by progesterone. In keep- ing with this interpretation, there is a marked reduction in the effectiveness of DTG in potentiating the NMDA response during late pregnancy (Bergeron et al., 1995b), a period during which progesterone levels are very high (Schwarz et al., 1989). Given the I very low doses of progesterone administered in the present study, 0 Control (female, day 3) 0 14 days following OVX one can assume that its effect is of physiological relevance. In conclusion, DHEA and progesterone appear to act as potent 0 DTG (1 pg/kg. I v.) El DTG (1 pg/kg, t.v.) modulators at g receptors. Because the prototypical (T ligand Progesterone (20 @g/kg, i.v.) Progesterone (20 Kg/kg, i.v.) q SKF-10047 produces marked neuropsychological perturbations in n Haloperidol (20 Kg/kg, i.v.) humans, the well known neuropsychological effects of neuroactive steroids in the course of the menstrual cycle or pregnancy might ,?g‘igl~e 8. 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