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MS 8716 Received 12 September 1998; accepted after revision 26 October 1998.
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ABSTRACT |
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-oestradiol (OE2), at a concentration as low as 0·1 nM, suppressed the magnitude of the resultant HFS-induced potentiation in slices from prepubertal animals (3 and 4 weeks old) of both sexes.
-oestradiol (OE1), progesterone (PRG) or testosterone (TST) on LTP in the young animals, even at a concentration 100 times greater than was effective for OE2.
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INTRODUCTION |
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One class of compounds that has long been known to have endocrine functions related to mammalian reproductive systems is the steroid hormones. Amongst this family of molecules, there is additional evidence that oestrogens are present within the central nervous system and can produce rapid effects on neuronal excitability (Kelly et al. 1980; Smith et al. 1987; Wong & Moss, 1994). The oestrogens are distributed heterogeneously and have been proposed to play a role as synaptic transmitters or neuromodulators (McEwen, 1991; Wong & Moss, 1992; Fahey et al. 1995).
Oestrogen exerts diverse modulatory effects on developmental processes, e.g. pubertal onset and neuroendocrine release (Ramirez & Sawyer, 1965; Smith & Davidson, 1968; Döcke et al. 1978; Kawakami et al. 1978). It has also been demonstrated to affect neuronal excitability (Kelly et al. 1977, 1980; Wong & Moss, 1992, 1994) and to influence certain aspects of learning and memory (Ball, 1926; van Haaren et al. 1988). For example, there is behavioural evidence that supports an involvement of oestrogens in suppressing (Zuckerman, 1952) or enhancing (Sherwin, 1988) performance on learning- and memory-related tasks. Silverman & Eals (1992) found that women who were in the menstruating phase of their cycles when oestrogen levels are low performed much better on a task involving mental rotation of a complex object than they did at other times, when oestrogen levels were higher. These and related functional observations in humans (Vázquez-Pereyra et al. 1995), plus other features of oestrogenic and related sex steroidal function in the neural and endocrine systems of developing (Morley et al. 1992) and mature (Isaacson et al. 1995) mammals, suggest that this steroid family has important roles in the modulation of brain mechanisms, particularly during ontogeny.
Whereas the induction of certain forms of long-term potentiation (LTP) in area CA1 of the rat hippocampus is known to involve NMDA receptors (Collingridge & Bliss, 1987), there have yet to be reported any studies on the possible pharmacological influence of gonadal steroids in modulating the input-output functions mediated in the hippocampus by the NMDA receptor during induction of synaptic plasticity (Smith, 1991). Even so, it is known that hippocampal dendritic spine density is regulated by OE2 via an NMDA receptor-dependent mechanism (Wooley & McEwen, 1994) and intracellular Ca2+ is increased during synaptic transmission by a modulatory action at the NMDA receptor in the presence of pregnenolone sulphate (Fahey et al. 1995). Such knowledge of a key role for oestrogenic substances in hippocampal plasticity would be valuable and potentially significant, given the wide variety of neuronal mechanisms that are affected by sex steroids in the normal development of the mammalian CNS (Ramirez & Sawyer, 1965; Smith & Davidson 1968; Greenstein et al. 1977; Morley et al. 1992), and in light of the inconsistencies in the data with oestrogen from behavioural studies, as referred to above. The present experiments were designed to examine possible alterations in the efficacy of synaptic transmission produced by oestrogen and related steroids in the hippocampus of developing and mature rats.
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METHODS |
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Chemicals and animals
All chemicals and drugs were reagent grade and were pretested by the manufacturer in cell culture for purity. OE1, OE2, PRG, TST and TMX were purchased from Sigma. They were dissolved according to the method of Smith et al. (1987) using 0·01 % propylene glycol-saline (165 mM NaCl). Wistar rats (Charles River), kept on a 12 h light/dark cycle, were used. A series of pilot experiments using adult females showed that there was no significant difference in the magnitude and duration of LTP expressed from slices taken from animals in the light and dark phases of their cycles, nor was there a difference in the lack of effectiveness of OE2 in altering LTP (see below) between the two light phases and so the data presented in this paper reflect only values obtained from experiments performed within the light phase of the cycle. Three groups of rats divided by age and sex were used. The 3-week-old animals were evenly divided into five females and five males; the 4-week-old animals were divided into seven females and five males and the 3-month-old animals comprised four females and five males. Prior to decapitation, mature females were subjected to a vaginal smear test. Every female was found to be in di-oestrous stage 1 of the oestrus cycle, i.e. cornified epithelial cells and leukocytes were present in the smear.
Slice preparation
All animals were killed by cervical dislocation followed by rapid decapitation after which the brains were removed and hemisected within a 2 min period. Following extraction of the hippocampal formation, slices of 400 µm thickness were cut on a rotoslicer (Dosaka EM; Ted Pella Inc., CA, USA) and were transferred to an incubation chamber containing artificial cerebrospinal fluid (ACSF) maintained at 30°C. The hippocampus was superfused constantly with ACSF during all phases of tissue extraction and incubation. This medium (pH 7·4) comprised (mM): NaCl, 124; KCl, 5·0; NaH2PO4, 1·25; MgSO4, 2·0; CaCl2, 2·0; NaHCO3, 22·0; glucose, 10·0; and was bubbled constantly with a gas mixture of 5 % CO2-95 % O2. Slices were incubated in this medium for at least 60 min prior to the first recording session.
Extracellular recording and stimulation
Slices to be tested were transferred from the incubation container to the superfusion chamber and placed between a supporting metal mesh stage and an overlying nylon mesh. The tissue was thus suspended about 100 µm below the surface of the oxygenated ACSF (infused at a rate of 3·0 ml min-1). A bipolar stimulating electrode was placed in the Schaffer collateral-commissural pathway (stratum radiatum) within the CA2 area. Glass micropipettes with impedances of less than 10 M
and filled with ACSF were used for the extracellular recording of population spike and field EPSP (fEPSP) responses in the pyramidal cell-body layer (stratum pyramidale) of area CA1.
For the recording of stimulus-evoked, population spike and fEPSP responses, a train of stimuli, each shock having a pulse width of 200 µs, was presented at a frequency of 0·1 Hz, at an intensity of less than 100 µA. Responses were observed on a storage oscilloscope and were detected aurally using an audio monitor. The population spike and fEPSP responses were recorded by an Axoclamp-2A (Axon Instruments) amplifier. The signals were transformed by an A/D converter (1401, Cambridge Instruments, Cambridge) and logged into a personal computer for additional on-line viewing and analysis.
Experimental procedures
Baseline population spike and fEPSP responses were elicited by adjusting the stimulus voltage to produce a half-maximal amplitude of the evoked responses; this intensity of stimulation remained invariant throughout the remainder of all phases of the experiment, including during high-frequency stimulation (HFS). Orthodromic stimuli of the same parameters as described above were delivered to the slice and stable baseline responses were recorded for at least 10 min prior to drug administration (these responses will henceforth be referred to as deriving from conditioning stimuli). After ensuring response stability, the delivery of normal ACSF ceased and the appropriate concentration of PRG, TST, OE1, OE2 alone, or OE2 together with tamoxifen (TMX), was administered. Turnover of the perfusion medium within the recording chamber from one solution to the next took place within 2 min. The concentration of TMX used was based on pharmacological and neurochemical data from studies with human breast cancer cells in vitro and rat uterine cytosol binding assays (Katzenellenbogen et al. 1983). Furthermore, Sutherland & Murphy (1982) showed that TMX at a concentration of 10-7 M is sufficient to antagonize the effects of oestrogen.
After approximately 10 min of perfusion with the drug-containing ACSF, a HFS (100 Hz for 1 s) was delivered to the slice. Thereafter, single shock stimuli resumed, using identical parameters as for the conditioning stimuli. Five minutes after the HFS, the delivery of ACSF was adjusted so that drug-free medium once more perfused the slice. For the control (drug-free) experiments, all conditions were the same except that no steroid entered the recording chamber. In some experiments, drug removal followed by a second application of HFS was performed. In these cases, normal ACSF was reperfused for 30-50 min after delivery of the first HFS. Following this 30-50 min period, a second sequence of HFS, applied using the same intensity as employed previously, was delivered to the slice to obtain a recovery sequence.
Whole-cell recording
Whole-cell recordings of CA1 neurones were performed blind on two groups of animals: the 3-week-old and the adult (3-month-old) rats. Eight slices from each of five animals were studied for both age groups. Slices 500 µm thick were used. The standard solution (pH 7·4) comprised (mM): NaCl, 124; KCl, 5·0; CaCl2, 2·5; MgCl2, 2·0; NaHCO3, 22·0; NaH2PO4, 1·25; glucose, 10·0; and was bubbled constantly with a gas mixture of 5 % CO2-95 % O2. Slices were incubated in this medium for at least 60 min prior to the first recording session. The medium used for slice perfusion was a low-Mg2+ solution, produced by replacing 2·0 mM with 0·5 mM MgCl2. This solution was modified further to isolate NMDA currents by the addition of tetraethylammonium chloride (TEA, 1 mM, Sigma), CNQX (10 µM, Tocris), (-)-bicuculline methiodide (BMI, 25 µM, RBI), and picrotoxin (PTX, 10 µM, Funakoshi, Tokyo, Japan). Additionally, when required, AP5 (50 µM, Tocris) or OE2 (10 nM) was added to the above. Patch pipettes were pulled from borosilicate glass capillaries (1·5 mm o.d., 1·12 mm i.d.; WPI, Sarasota, FL, USA) by a double stage puller (Narishige, Tokyo, Japan) so as to have resistances of 5-7 M and 1-2 µm tip diameters when filled with solution of the following composition (mM): caesium methanesulphonate, 120 (CH3O3SCs, Sigma); Hepes, 10 (Sigma); EGTA, 0·5; Mg-ATP, 2; MgCl2.6H2O, 1; TEA, 1; QX-314, 1 (N-(2,6-dimethylphenylcarbamoyl-methyl) triethylammonium bromide; RBI). The pH was adjusted to 7·3 with CsOH and the osmolarity was 285-300 mosmol l-1. The pipette series resistance was 20-35 M and was not compensated so as to maintain as high a signal-to-noise ratio as possible. The series and input resistances were monitored throughout the experiment and if either changed by > 15 %, the experiment was omitted from later analysis.
Synaptic responses evoked by the stimulating electrode were collected every 30 s using an Axopatch 1D (Axon Instruments) instrument. Unless otherwise noted, excitatory postsynaptic current (EPSC) was recorded at a holding potential of -80 mV in voltage clamp mode (filter setting, 1 kHz; digitized at 3-5 kHz on an A/D converter (Lab-PC+, National Instruments, Austin, TX, USA)) and stored on computer using custom-written software. To evaluate the effects of drugs on EPSC, the peak amplitudes of the EPSCs were measured continuously on-line and the results plotted on a cathode ray tube display.
Nature of the data and comparisons of groups
Two measures were sampled from the extracellular data set: population spike amplitude and the slope of the rising phase of the evoked fEPSP response. Both of these response variables were measured in the drug-free and drug-present conditions in order to evaluate the effects of each of OE1, OE2, PRG and TST alone, and of OE2 together with TMX. After six successive stimuli, the resultant (1) mean amplitude of the population spike responses, and (2) mean slope values of the fEPSP waveforms were calculated by computer, and the signal-averaged amplitude and waveform measurements were plotted on-line. The amplitude value was obtained by measuring the vertical distance between the peak negativity of the population spike response and the point of intersection with a line tangent to the two positive-going peaks on either side of the negative-going peak; the slope values for fEPSPs were obtained using conventional methods. This process continued during conditioning stimulation for at least 10 min. The mean amplitudes of the population spikes and fEPSPs elicited during this 10 min period were calculated. A HFS was delivered, and the post-HFS responses obtained were measured at regular intervals for 40 min or more following the last shock of the HFS. These data were expressed as a percentage of the mean response level during conditioning stimulation (% control). Whole-cell recordings were analysed following conventional methods.
Statistical treatment was with a two-way ANOVA, using values obtained 40 min following HFS. The main effect was the presence and absence of steroid (e.g. for OE2, F (1, 29) = 17·21, P > 0·0003), and age (4 weeks and 3 months; F (1, 29) = 21·20, P > 0·0001). Interaction effects were analysed using the parametric Student- Newman-Keuls procedure (Sokal & Rohlf, 1969) at a probability criterion of 0·05. Results of this test provided statistically significant main effects of the model, the data demonstrating a significant interaction between the two independent variables. Unless stated otherwise, n values refer to number of animals used in each group, not number of slices tested. The mean magnitude of LTP for each group was tested by post hoc tests which adjusted for unequal sample sizes.
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RESULTS |
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There were no differences in the magnitudes of LTP exhibited from slices taken from male and female animals. In the 3-week-old group, slices taken from males exhibited an increase in population spike amplitude following HFS of 292 ± 52 % and fEPSP slope of 209 ± 38 %, whereas slices taken from females showed a population spike increase of 278 ± 35 % and increased fEPSP slope of 195 ± 28 % (±1
Both LTP and STP were observed routinely following HFS at all ages examined whenever the slices were perfused with drug-free ACSF. STP, as measured by mean population spike amplitude, comprised 353 ± 51, 311 ± 35 and 237 ± 26 % of the control response for the 3 week (n = 10), 4 week (n = 12) and 3 month (n = 9) age groups, respectively. In terms of fEPSP slope, the mean values for the same age groups were 275 ± 43, 230 ± 28 and 168 ± 25 %, respectively. LTP, measured 40 min after HFS and expressed in terms of the mean increases in population spike amplitude, comprised 283 ± 41, 227 ± 24 and 182 ± 19 % of the control response while the increases in fEPSP slope were to 202 ± 30, 180 ± 28 and 145 ± 21 % of control for 3-week-, 4-week- and 3-month-old animals.
OE2 at a concentration of as low as 0·1 nM (as well as 10 nM) suppressed both STP and LTP in slices from the 3- and 4-week-old animals but not in the adult group (3-months old) from the levels seen in controls in a statistically significant manner (P < 0·01, Student-Newman-Keuls test), irrespective of whether population spike amplitude or fEPSP slope were analysed. Measurements of STP, made 1 min after HFS in the presence of OE2 (0·1 nM), showed the population response amplitudes for the 3 week, 4 week and 3 month age groups to be: 219 ± 47, 220 ± 41 and 248 ± 34 %, and the fEPSP slopes to be: 209 ± 42, 186 ± 23 and 180 ± 30 % of control, respectively. Measurements of LTP, made 40 min after HFS in the presence of OE2 (0·1 nM) yielded data for the population spike amplitude for the same age groups of: 182 ± 31, 132 ± 14 and 187 ± 26 %, and for the fEPSP slope of: 132 ± 22, 120 ± 15 and 138 ± 16 %, respectively. At the higher concentration of 10 nM, relatively greater decreases in both STP and LTP were obtained for population spike amplitude and fEPSP slope in the 3- and 4-week-old groups, but still no statistically significant difference was observed for the 3 month group, either upon induction of LTP, rapidity of onset, or maintenance; nor was any effect observed on amplitude of the population spike responses, slope of the EPSP wave or rate of decay during the 40 min period after HFS (n = 9).
Results from a single experiment with a 4-week-old female are shown in Fig. 1, where the effects of OE2 on the amplitudes of the population response and afferent fibre volley (FV), as well as fEPSP slope, both prior to and following HFS, are presented along with sample synaptic traces. In this example, a short-term synaptic enhancement of both postsynaptic measures is observed during the first 15-20 min post-HFS, but this decays to baseline levels before 40 min. No change was observed in amplitude of the FV with the addition of OE2, nor was there any change following HFS. Tests made with the solvent (0·01 % propylene glycol-saline) used to prepare the solutions of steroids on the two measures of plasticity showed that there was no effect on expression of LTP observed for as long as 40 min following HFS (n = 3).
Aa-d, population spike recordings; Ae-h, field EPSP slopes showing sample recordings taken prior to (a and b, and e and f) and after (c and d, and g and h) HFS, at times corresponding to similarly lettered points on the graphs in B and C. Each circle in B, and triangle in C, shows population spike (PS) and fibre volley (FV) amplitude data, and EPSP slope data, respectively, recorded during each minute of a trial, representing the computer-calculated means of 6 successively sampled sweeps. Crosses in B represent amplitude of FV. Hatched bar below graph indicates period of time during which OE2 was administered.
In Fig. 2 the main results are shown as means using data from 4-week-old animals and indicating the effects of the maximal and minimal concentrations of OE2 tested. The data with OE2 are presented together with the results from drug-free controls (squares). Each group consists of data from trials based on observations from 29 different slices. For the slices tested with OE2, 1 min after HFS the mean amplitude of the population responses had increased to 220 ± 41 % for 0·1 nM (circles), and to 163 ± 25 % for 10 nM (triangles). For the lower concentration, this potentiated amplitude diminished gradually until it plateaued at a value of from 136 ± 15 % (20 min) to 132 ± 14 % (40 min, difference not statistically significant). Somewhat lower but still comparable levels of change were observed when the fEPSP slope was assessed. The mean slope 1 min after HFS in the presence of OE2 (0·1 nM and 10 nM) reflected an increase to about 189 ± 17 % (circles) and 144 ± 19 % (triangles), respectively, and these values diminished rapidly (over the next 5 min) to asymptote by about t = 10 min. Tukey's post hoc tests performed for both population response amplitude and fEPSP slope showed the values at 40 min to differ significantly from the age-matched, drug-free controls (P < 0·01). For the 10 nM concentration of OE2, a nearly complete suppression of LTP was observed for both protocols (Tukey's post hoc comparison, P < 0·01) (Fig. 3). In an additional set of experiments on slices from 4-week-old animals (data not shown), OE2 (10 nM) administered for 10 min commencing 40 min after cessation of HFS, had no effect on the expression of LTP (215 ± 28 % for population spike; 172 ± 25 % for field EPSP, n = 4).
HFS was delivered at t = 0. Amplitudes of the population spike responses (A) and field EPSPs (B) are plotted against the ordinate scale where 100 % refers to the value of the response prior to HFS, irrespective of the test condition (
Summary of the effects of HFS on the magnitude of the increase of CA1 population spike responses (A) and field EPSPs (B) in the drug-free condition (open bars) and in the presence of OE2 (hatched bars: 0·1 nM; shaded bars: 10 nM), in the hippocampus of 3-week-old (n = 10), 4-week-old (n = 12) and 3-month-old (n = 9) rats. Results with males and females in all categories pooled. Error bars in this and all other figures represent ± 1
No difference in effectiveness of OE2 in the younger groups was found that could be attributed to sex (e.g. compare Fig. 4, data taken from a male, with Fig. 1, from a female). Note from Fig. 4 that a robust potentiation of the CA1 population response amplitude and fEPSP slope was elicited by a second HFS applied to this slice, approximately 35 min after removal of OE2 from the perfusion chamber. Similar results were obtained from the other four males studied from this age group.
A, population spike data; B, field EPSP data. Two series of HFS were delivered to the same slice, the 1st in the presence of 10 nM OE2, and the 2nd 40 min later (33 min after removal of OE2 from the perfusing medium). For all other conventions, see legend to Fig. 1.
To investigate the hormonal specificity of action of OE2, its effects were compared with those of two other sex-related steroid hormones, PRG and TST, as well as with OE1, the inactive analogue of OE2, at 10 nM, the concentration that produced the strongest OE2-induced suppression. Pharmacological (see references in Smith, 1991) and neurochemical (Towle & Sze, 1983) experiments have shown PRG and TST to be of similar potency to OE2 at central neurones. Neither PRG nor TST in any way produced disturbances in the induction or maintenance of LTP in slices from any of the three age groups tested, nor were there any sex-related differences noted in this lack of effect. In 12 slices from 4-week-old animals, measures of mean population spike and fEPSP slope following HFS expressed as percentages of drug-free conditioning amplitudes were 227 ± 24 and 180 ± 28 % (respectively) in drug-free controls; 104 ± 8 and 101 ± 12 % in the presence of OE2 (n = 12, difference statistically significant from corresponding measures in drug-free medium, Student-Newman-Keuls post hoc test, P < 0·01); 180 ± 42 and 170 ± 33 % in the presence of TST (n = 6); and 196 ± 34 and 167 ± 31 % in the presence of PRG (n = 6) (all drugs perfused at 10 nM; see Fig. 5A and B). OE1 was tested on an additional set of three slices from the 4-week-old group; no action consistent with a suppression of synaptic transmission, nor of suppression of HFS-induced LTP such as was obtained with OE2, was observed (data not illustrated).
Examples of the results of tests with PRG (A) and TST (B), both delivered at 10 nM, on LTP in slices from 4-week-old rats. Each substance was administered 10 min prior to HFS and was removed from the perfusing medium 10 min following cessation of stimulation.
It was considered of interest to attempt to determine whether the effects observed with OE2 on LTP suppression were likely to be mediated by receptors on the cytoplasmic membrane. Our pharmacological approach to this question involved the use of TMX, an anti-oestrogen compound having an action at cytosolic (i.e. intracellular) receptors. An example is given in the traces of Fig. 6. When delivered alone to slices from 4-week-old animals, TMX (1·0 µM, n = 4; 10 µM, n = 6) alone exerted small, non-significant effects in 6 of 10 tests (decreases in 4 and increases in 2 tests, respectively; no effect in 4 cases), on the amplitude of the population response and fEPSP slope (Fig. 6B). There also was no effect of the antagonist on the establishment of LTP (data not shown). When the antagonist was delivered at either concentration in combination with 10 nM OE2 (n = 7), TMX in all cases was found to be ineffective in preventing the OE2-induced suppression of LTP (population spike amplitude 40 min after HFS = 110 ± 8 %; fEPSP slope = 106 ± 8 %. After a period of perfusion with normal (drug-free) ACSF of at least 30 min to eliminate the drugs used from the recording chamber, subsequent HFS applied to the same slices elicited robust LTP (e.g. Fig. 6Ad and Bd).
A, sample synaptic traces and, B, graphical representation of data from a single experiment illustrating lack of antagonism by TMX (administered during period shown by hatched bar) of the OE2-induced (filled bar) suppression of LTP in area CA1. a, drug-free condition; b, 60 min after administration of TMX (1·0 µM); c, 40 min after HFS in the presence of OE2 and TMX and 25 min following drug removal; d, 30 min after a second HFS delivered during drug-free condition. In A, top, sample traces of population spike data; A, bottom, field EPSP traces. For all other conventions, see legend to Fig. 1.
To examine whether the HFS-induced LTP was NMDA receptor dependent prior to performing tests related to attempting to determine the possible mechanism of action of OE2, AP5 was tested on slices from 3-week- and 3-month-old animals. Population spikes and fEPSPs were recorded extracellularly in the pyramidal cell layer and stratum radiatum, respectively, using six slices from each age group. HFS induced LTP of population spike amplitude in young (270 ± 35 %) and adult (189 ± 21 %) originals, and of fEPSP slope in young (183 ± 33 %) and adult (151 ± 24 %) animals. Perfusion with AP5-containing medium (50 µM) for 10 min prior to HFS completely suppressed LTP of both population response amplitude (107 ± 25 %) and fEPSP slope (102 ± 23 %) in young animals, and population response amplitude (101 ± 18 %) and fEPSP slope 103 ± 14 %) in adults, respectively, in a statistically significant manner (Student's t test, P < 0·01).
Whole-cell recordings from CA1 cells yielded input resistances of 94-147 M
A, from the 3-week-old group. B, from the 3-month-old group. Aa, control response. Ab, in the presence of CNQX (10 µM), BMI (10 µM) and PTX (10 µM) (CBP solution). Ac, in the presence of the same drugs as Ab but with AP5 (50 µM) added. Ad, the response in CBP solution after removal of AP5. Ae, after addition of OE2 (10 nM). Af, recovery after removal of OE2 in CBP solution. Ba'-e', same conventions as for Aa-f, but recordings from a neurone in an adult. Vertical bar is 60 pA for a' and 30 pA for b'-e'. C, bar histograms showing data relating only to the isolated, AP5-sensitive component of the synaptically elicited responses of young and adult CA1 neurones. Left pair of bars represent this component in young (3 weeks, hatched column, n = 8) and adult animals (3 months, shaded column, n = 8). Right pair of bars represent the OE2-sensitive component in the two age groups, as indicated. Note the much greater OE2-sensitive component in the responses from the young neurones. ** Statistically significant (Student's t test, P < 0·01).
In order to evaluate the effects of OE2 on the AP5-sensitive component of the synaptic response, administration of OE2 (10 nM) caused a strong suppression of the currents from young neurones but considerably less suppression from adult neurones (Fig. 7Ae and Be'), this effect appearing within about 5-7 min after onset of perfusion and the effect requiring about 4 min to reach plateau. The OE2-sensitive component of the response (labelled OE2 in Fig. 7C) was 50·7 ± 13·2 % (n = 8) of the response in CBP solution for the young neurones whilst that of the adult neurones was 3·4 ± 6·2 % (n = 8) of the response in CBP solution (right pair of bars, Fig. 7C). The difference between these values is statistically significant (Student's t test, P < 0·01).
Many cellular actions characteristic of the steroid hormones, especially in non-neural tissue, are known to begin by the hormone first diffusing across the cytoplasmic membrane of the target cell to bind with intracellularly located receptors, after which the resultant complex translocates to the nucleus and a cascade of events takes place that includes chromatin binding, modification of gene transcription, polypeptide synthesis and production of a change stereotypical for the cell affected. This relatively long-term series of events takes in the order of hours to complete and is known as the genomic mechanism of action (Schrader et al. 1980; Morley et al. 1992). In neural tissue, non-genomic mechanisms have been described which are thought to involve rapid time courses (seconds) that are mediated by surface-bound receptors, similar to the actions of the more classical synaptic transmitters (Kelly et al. 1977, 1980; Wong & Moss, 1992; for review, see Orchinik & Murray, 1994). Those studies have shown that the extracellular administration of steroids, such as OE2, exerts changes in patterns of firing and of membrane potential in cells from many areas of the brain, including hippocampus (e.g. Joëls & Karst, 1995), within one or just a few seconds. The rapid cessation of effect and reversibility of the response elicited argues for an action whereby the steroid associates and dissociates quite readily from the external surface of the neural membrane (Orchinik & Murray, 1994). This time course, and that seen in the present study for the actions of OE2 on LTP, is consistent with the idea that the steroid may exert direct effects on external receptors involved in synaptic transmission, or in its moment-to-moment modulation (Isaacson et al. 1995).
The ineffectiveness of TMX in antagonising the OE2-mediated response studied here supports further the suggestion that the actions of the steroid are likely to have taken place at the level of the cell membrane, since TMX is thought to act exclusively at intracellular sites through a block of the genomic mechanism (Wakeling, 1995). Smith et al. (1987) demonstrated that TMX did not block OE2-induced potentiation of glutamate-induced excitations in the cerebellum, leading to the proposal there that the steroid-induced enhancements which they observed were due to a rapid action at the level of the cellular membrane.
Loy et al. (1988) examined the distribution of OE2 and TST binding in the hippocampal formations of male and female rats in an autoradiographical study. Neurones binding OE2 were located in the stratum pyramidale. It was hypothesized that these neurones might be sensitive to the effects of OE2 and that the cells may correspond to early-maturing pyramidal neurones. It was demonstrated also that there was no significant binding of TST anywhere in the hippocampal formation. Warembourg (1978) also obtained negative evidence with respect to binding of PRG in the hippocampus. These observations are in accordance with the present findings with hormone superfusion, that of TST, PRG, OE1 and OE2, only OE2 influences the magnitude of LTP expressed following tetanus, and that it may do so in a dose-dependent manner, implicating a receptor-mediated action as being responsible for the effect. In other systems where PRG and TST are known to have effects, their potencies as compared with OE2 have been shown to be of a similar order of magnitude (Towle & Sze, 1983; Smith, 1991). Further support for the view that the effects seen here were likely to be receptor mediated is found in the observation that the pharmacologically inactive form of oestrogen, OE1, also had no LTP-suppressant activity.
Two young age groups were examined in the present extracellular recording part of this study, 18- and 19-day-old (the 3 week group) and 4-week-old animals, to determine whether a change in effectiveness of OE2 against LTP might be apparent since OE2 did not affect LTP in tissue taken from mature animals. Although animals from the youngest group exhibited relatively greater levels of LTP than in the older groups, the effects of OE2 between the two immature groups did not differ significantly.
Several considerations relate to the interpretation of the present data. Firstly, during early postnatal life in rats, elevated levels of plasma
One possibility that may account for some of the age-related differences in the ability of the hippocampus to support synaptic plasticity could be that the relatively immature synaptic organization of the younger hippocampus, combined with the inevitably more labile evoked responses obtained there, contributes to the greater effectiveness of OE2 in suppressing LTP. Warren et al. (1995) showed that the magnitude of LTP in proestrous was greater than in oestrous. This result might derive in part from two possibly related factors, that is, of a greater number of spines and synapses seen in the animals during the former stage, coupled with the higher concentration of oestrogens during proestrous, possibly being responsible for the increase in spine and synapse number (Gould et al. 1990; Woolley & McEwen, 1992).
Relationship with amino acid receptors
Alfsen (1983) has shown that one result of steroid-protein interactions can be a conformational change that could provide a mechanism for the OE2-NMDA receptor interaction proposed here. In the brainstem for example, PRG and its metabolites react with membrane-bound proteins on neurones (Ke & Ramirez, 1990). PRG binding modulates voltage-sensitive calcium channels to produce a rapid influx of Ca2+, whilst it also has been demonstrated that 3-
It is additionally possible that OE2 interacts indirectly with the transmitter, as OE2 allosterically modulates glutamate dehydrogenase (Pons et al. 1978). The direction of effect (enhancement or suppression of enzyme activity) is concentration dependent; OE2 alters the activity of this enzyme differently when high concentrations of the substrate are present, such as would occur during HFS. Consequently, the presence of the steroid in conditions of high levels of glutamate enhance the binding of OE2 with glutamate dehydrogenase, thus diminishing the availability of glutamate at the NMDA receptor. For this reason as well, the present results should not be considered in conflict with the data of Wong & Moss (1992, 1994) who showed EPSP-enhancing effects and increases in excitability with OE2 using protocols that result in shorter-duration synaptic activation and lower levels of synaptic release of glutamate than occur with HFS.
The role that OE2 might play in higher-order mental functions such as learning or memory has not yet been resolved from the behavioural literature, since many of the accounts that exist contain mutually inconsistent findings about the direction of effect of OE2 on cognitive processes (Ball, 1926; Zuckerman, 1952; van Haaren et al. 1988; Sherwin, 1988; Vázquez-Pereyra et al. 1995). The present findings with hippocampal LTP might be interpreted as suggesting that elevated levels of OE2 in the period of development prior to the onset of puberty may exert a negative influence on neuronal events associated with use-dependent aspects of synaptic transmission. To submit this conjecture to critical evaluation, it would be interesting to examine further the effects of OE2 in prepubertal animals during the performance of appropriate behavioural tasks.
This study was supported by a grant from the Human Frontier Science Programme (to T. P. H.), an intramural award from the UNCG Research Council, and funds provided by the NRC (Canada). We thank Dr Sheryl S. Smith (Philadelphia) for her many helpful suggestions and encouragement.
Corresponding author
T. P. Hicks: Neural Plasticity and Regeneration Group, Institute for Biological Sciences, National Research Council of Canada, Building M-54, 1200 Montreal Road, Ottawa, Ontario, Canada K1A 0R6.
Email: philip.hicks{at}nrc.ca
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Figure 1. Data from a single experiment, using a hippocampal slice from a 4-week-old female in the presence of 10 nM OE2 during administration of HFS
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Figure 2. Mean effects of OE2 (0·1 nM and 10 nM) on elicitation of LTP from area CA1 in slices from 4-week-old rats
, drug free; 
, 0·1 nM OE2; and 
, 10 nM OE2). Points in each of the 3 conditions represent means of 12 observations.
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Figure 3
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Figure 4. Effect of OE2 on LTP in a slice taken from the hippocampus of a 4-week-old male
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Figure 5
, population spike data; , field EPSP data.
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Figure 6
(n = 8) in slices from young animals and 107-168 M (n = 8) in slices from adults. In standard media, during voltage clamp at -80 mV, inward synaptic current was recorded following activation of afferent fibres at 30 s intervals as shown in Fig. 7A and B. Slightly increasing stimulus intensity produced larger EPSCs of similar shape. The range of EPSC peak amplitudes was 83 to 361 pA (n = 8) in young (Fig. 7A) and 63 to 340 pA (n = 8) in adult (Fig. 7B) neurones. EPSC amplitude decreased after administration of a mixture of CNQX (10 µM), BMI (25 µM) and PTX (10 µM) (CBP solution) until after about 15-20 min it reached a plateau level, resulting in what then could be considered the NMDA current (Fig. 7Ab and Bb'). The CNQX-sensitive component (probably representing AMPA current) was calculated by subtraction of the response in CBP solution from that in standard solution. To determine whether the response in the presence of CBP solution was current mediated through the NMDA channel, AP5 was added to the CBP solution. Virtually the entire synaptic response was abolished in both young and adult neurones (Fig. 7Ac and Bc', respectively). The AP5-sensitive component was 88·3 ± 7·4 % (n = 8) of the remaining response when recordings were made in CBP solution in young cells, and 85·6 ± 9·3 % in adult cells (left bars in Fig. 7C, labelled AP5). The NMDA component was calculated from the subtraction of the response in the CBP/AP5-containing medium (Fig. 7Ac and Bc') from that in CBP medium (Fig. 7Ab and Bb'). The ratio of the NMDA component to the AMPA component for the young neurones was 32·1 ± 10·8 % (n = 8) and for the adult neurones was 16·5 ± 7·4 % (n = 8). The difference between these values is statistically significant (Student's t test, P < 0·01). This result suggests that the NMDA component of the excitatory synaptic response of young CA1 neurones is relatively larger than that seen with adult neurones, notwithstanding that it is not certain whether the absolute number of NMDA receptors in the young CA1 region is greater than in the adult. The synaptic response recovered 20-30 min after removal of AP5 from the perfusing solution (Fig. 7Ad and Bd').
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Figure 7. The effect of OE2 on synaptic responses recorded in whole-cell mode showing the component mediated through NMDA receptors in two neurones
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-fetoprotein protect the developing brain from circulating OE2 by binding preferentially to this steroid (Raynaud et al. 1971). Until about the 20th postnatal day, more than 99 % of plasma-located OE2 is so bound (Greenstein et al. 1977). Within the subsequent 8 days, however, -fetoprotein disappears from the blood and unbound OE2 becomes physiologically active. During this time, serum levels of gonadotropin and OE2 decrease substantially (Döhler & Wuttke, 1975). This critical transitional period in postnatal life demarcates the onset of puberty, which usually begins within the 5th week. Administration of OE2 has been found to advance the onset of puberty; induction has occurred as early as the 22nd day (Ramirez & Sawyer, 1965; Smith & Davidson, 1968; Döcke et al. 1978). It might be expected therefore that serum levels of OE2 would increase slightly, at least, just prior to the onset of puberty; however, instead, tissue levels increase at a time that coincides with onset of puberty (Döhler & Wuttke, 1975). Our results with 3- and 4-week-old animals suggest that administration of OE2 to naïve, or unprimed tissues may activate receptor-mediated processes accompanying onset of puberty. Indeed, Kawakami et al. (1978) have suggested that OE2 is involved in some form of reorganization of the hippocampus, one result of which may be to stimulate the induction of control mechanisms responsible for the timing of pubertal onset.
-hydroxy, 5--pregnan-20-one (3-OH-DHP), a metabolite of PRG, binds to a component of the GABAA receptor complex, allowing the Cl- channel coupled to the complex to remain open for longer than normal (Lan et al. 1990). Wooley & McEwen (1994) have shown that oestrogen appears to modify dendritic spine density through an action directly at the NMDA receptor and then only during specific times during the oestrus cycle when circulating levels are high. Wong & Moss (1992) reported that in a subpopulation of CA1 pyramidal cells, treatment with OE2 results in a prolongation of the EPSP and a period of increased probability of repetitive firing in response to stimulation of synaptic input from the Schaffer collateral-commissural system. The sex hormone precursor pregnenolone sulphate potentiates NMDA receptor-mediated increases in intracellular Ca2+ in young animals by way of positive allosteric modulation of the receptor and does not interact with the polyamine modulatory site (Fahey et al. 1995). These observations with sex hormones and related sex steroids provide evidence to support the view that OE2 might react with receptors to effect rapid modulatory events at NMDA binding sites in young animals to influence processes of synaptic transmission at the level of the cell membrane, and thereby influence synaptic plasticity. Our results using whole-cell recordings are consistent with this view. Further support in favour of this view are the recent findings of Weaver et al. (1997), who reported that OE2 protects against NMDA-induced excitotoxicity through a direct inhibitory action on the NMDA current in cultured embryonic rat hippocampal cells.
REFERENCES
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