Introduction

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Activation of corticosteroid receptors in the hippocampus influences hippocampus-related behaviors (de Kloet et al., 1993) and modulates the activity of the HPA axis, which regulates the synthesis and secretion of corticosteroids (Jacobson and Sapolsky, 1991). Corticosteroid hormones (glucocorticoids and mineralocorticoids) regulate gene expression through the interaction with two receptor subtypes: MR (or type I) and GR (or type II) (Joels et al., 1994). MR and GR have a 5- to 10-fold difference in their affinity for the rat corticosteroid hormone corticosterone. MR has a high affinity for corticosterone and is 70 to 80% occupied at basal corticosterone plasma levels (Krozowski and Funder, 1983; Reul and de Kloet, 1985; Reul et al., 1987a). GR has a lower affinity for corticosterone and its occupancy changes from 15% at basal to 90% at peak circadian or stress-induced circulating corticosterone levels (Reul and de Kloet, 1985; Reul et al., 1987a,b).

One way corticosteroids alter neural activity in the hippocampus is by modulating the responses elicited by the activation of neurotransmitter receptors (McEwen, 1996; Joels and de Kloet, 1994), including the serotonergic 5-HT_1A receptor (Joels and de Kloet, 1994; Beck et al., 1996). Several laboratories have provided detailed pharmacological characterization of the 5-HT_1A receptor in the CA1 and CA3 subfields of the hippocampus (Andrade et al., 1986; Andrade and Nicoll, 1987; Beck et al., 1992). The 5-HT_1A receptor is a G protein-coupled receptor. Activation of the 5-HT_1A receptor in the CA1 and CA3 hippocampal subfields increases an inward rectifying potassium current linked to a PTX-sensitive G protein (Andrade et al., 1986; Andrade and Nicoll, 1987; Colino and Halliwell, 1987; Beck et al., 1992; Beck and Choi, 1991; Okuhara and Beck, 1994). Corticosteroids alter the 5-HT_1A receptor-mediated responses in hippocampal subfield CA1 (Beck et al., 1996; Joels et al., 1991; Joels and de Kloet, 1992). Even though the 5-HT_1A receptor mediates a hyperpolarization through an increase in conductance of an inward rectifying potassium channel in both area CA1 and CA3, there are important differences in the nature of the response (Beck et al., 1992; Okuhara and Beck, 1994). For example the rank order potency of the agonists are the same, but the absolute affinity of the ligands are approximately a half of a log unit lower in affinity in CA3. The antagonists spiperone and BMY 7378 are competitive antagonists in CA1, but insurmountable in CA3. The effects of corticosteroids on the 5-HT_1A receptor-mediated increase in potassium conductance in subfield CA3 are not known; we propose that the effects of corticosterone on the 5-HT_1A receptor-mediated response will be different in subfields CA1 and CA3.

Previous studies have examined the effects of corticosteroids on the number of 5-HT_1A binding sites and mRNA levels in the hippocampus (Chaouloff, 1995). Corticosteroids appear to alter 5-HT_1A receptor binding sites in subfield CA3, but not in subfield CA1 (Mendelson and McEwen, 1992a,b; Kuroda et al., 1994). Recently, we reported that chronic exposure to high levels of corticosteroids increased the protein levels of G_s, G_i1and2 and G_o -subunits in the hippocampus (Okuhara et al., 1997). However, the actions of corticosteroids on G protein function in the individual hippocampal subfields and on G protein-evoked currents are not known.

In our study, we examined the effects of chronic corticosteroid treatment on different components of the 5-HT_1A receptor-effector pathway in hippocampal subfield CA3 pyramidal cells. Single-electrode voltage clamp techniques were used in the brain slice preparation. We hypothesized that chronic corticosteroid treatment would alter the 5-HT_1A receptor signal transduction system in subfield CA3. The results from these experiments will contribute to a basic understanding of how corticosteroids alter responses elicited by the activation of G protein-linked neurotransmitter receptors and help to elucidate the role of corticosteroids in hippocampus-related behaviors and HPA feedback.

	Methods

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Animals. Four different treatment groups were used for the experiments in this investigation (table 1). The adrenalectomy was performed as described previously (Beck et al., 1994; Birnstiel and Beck, 1995). Male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) 75 to 100 g were used for all treatment groups. Bilateral adrenalectomies were performed on all treatment groups, except the SHAM group, to remove circulating corticosteroids. The animal was anesthetized with ether, a small incision was made into the abdominal cavity, just below the rib cage, and the adrenal glands carefully removed. The muscle wall was then sutured and the skin closed using wound clips. The ADX group received no further treatment. The ALD group had an osmotic minipump containing aldosterone (Alzet model 2002 from Alza Cooperation, Palo Alto, CA), implanted s.c. at the time of adrenalectomy, to selectively activate MR. The minipump delivered 10 µg/hr aldosterone (Steraloids, Inc., Wilton, NH) dissolved in propylene glycol. MR and GR were activated in another group of animals (HCT) by s.c. implanting 200 to 300 mg corticosterone pellets, 2- to 3-wk release (Innovative Research, Toledo, OH), in the back of the neck at the time of adrenalectomy. The SHAM group of animals was produced by visualizing the adrenal glands but leaving them intact. After surgery the animals were maintained on a standard 12-hr light/dark cycle and rat food, ad libitum. SHAM animals were given standard drinking water while the ADX, ALD and HCT animals were given 0.9% NaCl drinking water ad libitum. At the end of 13 to 15 days, the animals were killed in the morning and hippocampal slices immediately prepared for electrophysiological recording. At the time of death, trunk blood was collected to determine the plasma corticosterone levels by radioimmune assay (Burgess and Handa, 1992).

Hippocampal slice preparation. Hippocampal slices were prepared for electrophysiological recording as previously described (Okuhara and Beck, 1994). Rats were killed by decapitation and the brain rapidly removed and placed in ice cold ACSF containing in mM: NaCl 125, KCl 3, NaH₂PO₄ 1.25, MgSO₄ 2, CaCl₂ 2.5, dextrose 10 and NaHCO₃ 28. The ACSF was also supplemented with steroids, as outlined in table 1, to maintain the treatment paradigm. We previously reported that steroids must be present in the ACSF to preserve the corticosteroid-induced effects on neuron cell properties (Beck et al., 1994). The hippocampus was dissected free and the dorsal section cut in 500- to 550-µm sections on a vibratome. The hippocampal slices were then placed in a holding vial containing ACSF bubbled with 95% O₂-5% CO₂ at room temperature. The slices remained in the holding vial for at least 1 hr after dissection before being transferred to the recording chamber. In the recording chamber, the slice was stabilized between two nylon nets and continuously perfused with ACSF bubbled with 95% O₂-5% CO₂ at a rate of 2 to 3 ml/min, at 31 to 32°C.

Intracellular recording. Intracellular recordings were made as previously described (Okuhara and Beck, 1994). Electrodes were pulled from borosilicilate capillary tubing on a Brown and Flaming electrode puller (Sutter Instruments, Novato, CA) to a resistance of 30 to 35 M (2 M KCl). Pyramidal cells were impaled with brief ejections of positive current through the electrode. The impaled cells were sealed by applying 1 nA hyperpolarizing current. Electrical signals were collected and amplified using an Axoclamp 2A and Cyberamp 320 amplifier (Axon Instruments, Foster City, CA) and recorded on a Gould Series 3200 chart recorder (Gould Electronics, Valley View, OH). Data were collected on-line with pCLAMP software (Axon Instruments).

5-HT concentration-response. Cells were voltage clamped at 65 mV and data for the 5-HT concentration response curves were collected by perfusing the slice with increasing 5-HT (5-HT hydrochloride, Sigma) concentrations (3, 10, 30 and 100 or 110 µM) and the maximum current response evoked during the drug application was recorded. During drug perfusion, if the voltage clamp deviated by more than 1 mV, the data were discarded.

Experiments with GTPS. The effect of corticosteroids on G protein activity was determined by measuring the magnitude of outward current evoked by 15 mM GTPS (Boerhinger Mannheim, Indianapolis, IN) which was included in the recording electrode. After the cell's membrane potential stabilized, the cell was voltage clamped at its resting membrane potential (i.e., the potential where no current was required to maintain the voltage clamp). The voltage clamp was then moved to 60 mV and the amount of current required to maintain the clamp was recorded.

I-V relationship. The effect of corticosteroids on the potassium current linked to the 5-HT_1A receptor was determined by plotting the current-voltage (I-V) curve for the outward current evoked by the activation of the 5-HT_1A receptor. After the cell's membrane potential stabilized, the cell was voltage clamped at its resting membrane potential (the potential where no current was required to maintain the voltage clamp). Data for I-V plots were obtained by running voltage ramps in the presence and absence of 100 µM 5-HT. Voltage ramps were run from 60 mV to +35 mV from the cell's clamped membrane potential, i.e., approximately 120 to 25 mV, at a rate of 1 mV/sec. The I-V plot for the potassium current evoked by the activation of the 5-HT_1A receptor was constructed by subtracting the control ramp values (no 5-HT) from the ramp values in the presence of 100 µM 5-HT. The reversal potential, conductance (between 105 to 60 mV) and potential where inward rectification occurred were analyzed for each curve.

Statistical analysis. Statistical comparisons were performed using ANOVA. The Student-Newman-Keuls method was used for post hoc tests. All values are reported as mean ± S.E.M. A P < .05 was considered significant.

	Results

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Corticosterone plasma levels. The mean corticosterone plasma levels for the different treatment groups are listed in table 1. SHAM corticosterone plasma levels ranged from 0 to 4.13 µg/dl. ADX corticosterone plasma levels ranged from 0 to 0.22 µg/dl. ALD corticosterone plasma levels ranged from 0 to 0.56 µg/dl. The lower and upper limits of the corticosterone radioimmune assay were 0.05 and 50 µg/dl, respectively. Adrenalectomies that produced corticosterone concentrations 0.6 µg/dl were considered successful. Two SHAM, 8 ADX and 14 ALD animals had corticosterone levels less than 0.05 µg/dl. The concentration of aldosterone used in our experiments was based on an investigation by Kuroda et al. (1994). The ALD treatment decreased MR binding by 61% (n = 5 animals) in the cytoplasm (data not shown) as determined using a homogenate binding assay. HCT plasma concentrations of more than 20 µg/dl were considered successful. HCT corticosterone plasma levels ranged from 25 to >50 µg/dl.

5-HT concentration response curve characteristics for the 5-HT_1A receptor. Activation of the 5-HT_1A receptor evoked an outward current in a concentration-dependent manner (fig. 1A). The 5-HT concentration-response curve characteristics recorded from cells from corticosterone treated animals are summarized in table 2. The EC₅₀ obtained in cells from ADX was smaller compared to cells from either SHAM-, ALD- or HCT-treated animals (fig. 1B). The E_max value was also smaller in cells from HCT when compared to cells from SHAM- or ADX-treated animals (fig. 1B). Corticosterone treatment did not alter the slope index.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent response of subfield CA3 pyramidal cells to 5-HT perfusion. A, Chart recording of the concentration-dependent current response of a CA3 cell to 5-HT perfusion. The drug was applied for the duration depicted by the line above the chart record. B, Concentration-response curves from data collected from all of the cells from SHAM- (open circles), ADX- (closed circles) and HCT- (closed squares) treated animals. The data were fit to a hyperbolic function as described in "Materials and Methods."

View larger version (11K): [in this window] [in a new window]   Fig. 2.   The outward current elicited by the perfusion of 30 µM 5-HT in the presence and absence of 1 µM of the 5-HT4 and 5-HT7 receptor antagonists GR113808 and ritanserin. The drugs were applied for the duration depicted by the line above the chart record. The magnitude of the outward current evoked by 5-HT was not different (P = .141, n = 6, paired t test) in the presence or absence of GR113808 and ritanserin.

Activation of G proteins with GTPS. The injection of GTPS into neurons produced a hyperpolarization of the resting membrane potential. There was no difference in the resting membrane potential between treatment groups. The resting membrane potentials were SHAM 76 ± 1 mV, ADX 79 ± 2 mV, ALD 74 ± 1 mV and HCT 76 ± 1 mV). The relative amount of current evoked by the activation of G proteins with GTPS was determined by voltage clamping the cell's membrane at its resting potential (when zero current is required to maintain the voltage clamp). The voltage clamp was then moved to 60 mV for several minutes (fig. 3A). Although holding the cell at 60 mV, a saturating concentration of 5-HT (100 µM) was applied; the response to 5-HT was totally occluded when GTPS was included in the recording pipette (fig. 3A). The amount of current required to maintain the voltage clamp at 60 mV was used as a measure of the amount of current evoked by the activation of G proteins with GTPS. Figure 3B summarizes the results of these experiments. Significantly less current was required in cells from HCT when compared to cells from SHAM-, ADX- and ALD-treated animals. The amount of current measured in cells from SHAM was also smaller compared to cells from ADX-treated animals.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   The outward current evoked by GTPS. A, Chart recording of a cell injected with GTPS. The cell's membrane potential was first voltage clamped at its resting potential (74 mV, I = 0 pA). The clamp was then moved to 60 mV and the current recorded (I = 340 pA). Perfusion with 100 µM 5-HT (duration depicted by the line above the chart record) did not elicit a current response, indicating all the G proteins linked to 5-HT receptors were activated by GTPS. B, Summary graph depicting the amount of current evoked by GTPS in cells from corticosterone-treated animals. Treatment group and number of cells are labeled at the bottom axis. ANOVA F = 12.691; d.f. = 3, 61; P < .001. Student-Newman-Keuls, P < .05; *HCT different from SHAM, ADX and ALD; #ADX different from SHAM.

I-V relationship. I-V plots for the 5-HT_1A evoked potassium current were analyzed in three to five cells from ADX-, SHAM- and HCT-treated animals. Representative I-V plots in figure 4A-C demonstrate that corticosterone does not appear to alter the reversal potential nor the inward rectification properties for the potassium current linked to the 5-HT_1A receptor. The reversal potential was between 97 to 104 mV and rectification started at approximately 50 mV. There was no significant change in the slope conductance (table 2) measured in the linear portion of the I-V plot, between 105 and 60 mV.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   I-V plots of steady-state 5-HT mediated current in a cell from A, SHAM-treated animal; B, HCT-treated animal and C, ADX-treated animal.

	Discussion

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The 5-HT_1A receptor is a member of the G protein-linked receptor family. In our study, we report the effects of chronic corticosteroid treatment on different components of the 5-HT_1A receptor-G protein-ion channel system in hippocampal subfield CA3 pyramidal cells using electrophysiological techniques. The EC₅₀ value for the 5-HT concentration-response curve was shifted to the left in cells from ADX-treated animals compared to SHAM, ALD and HCT. The E_max value was smaller in cells from HCT animals compared to ADX and SHAM. The shift in the 5-HT_1A response may be due to alterations at sites past the 5-HT_1A receptor. Therefore, we compared the magnitude of the outward current evoked by the activation of G proteins with GTPS. Less current was evoked by GTPS in cells from HCT animals compared to ADX, SHAM and ALD. Furthermore, less current was evoked by GTPS in cells from SHAM compared to ADX animals. Finally, we determined the effects of corticosteroids on the I-V relationship of the potassium current elicited by the activation of the 5-HT_1A receptor. Corticosteroid treatment did not alter the reversal potential, conductance or inward rectification properties of the potassium current linked to the 5-HT_1A receptor. Based on our results and those previously reported by other investigators, we conclude that corticosteroids alter the response elicited by the activation of 5-HT_1A receptors by modulating several components of the receptor-effector cascade.

The natural neurotransmitter, 5-HT, was used in these experiments to generate the concentration-response information. If corticosterone interfered with uptake mechanisms, it is possible this would result in an alteration in the 5-HT concentration-response curve for the 5-HT_1A receptor-mediated increase in outward current. However, it has been previously demonstrated that blocking the 5-HT uptake mechanisms with fluoxetine did not alter the characteristics of the 5-HT concentration-response curve for the 5-HT_1A receptor-mediated hyperpolarization (Beck et al., 1992).

Alterations in the number of 5-HT_1A binding sites may account for some of the changes we observed in the 5-HT concentration-response curve characteristics. Previous investigators reported that adrenalectomy increases the number of 5-HT_1A receptor binding sites in subfield CA3 when compared to either SHAM, aldosterone, low or high corticosterone-treated animals (Mendelson and McEwen, 1992a, 1992b; Kuroda et al., 1994; Chalmers et al., 1993; Tejani-Butt and Labow, 1994). The increased number of 5-HT_1A receptor binding sites may account for the shift to the left of the EC₅₀, with no change in E_max, for the 5-HT concentration-response curve that we observed in cells from ADX compared to SHAM- and ALD-treated animals. A shift in EC₅₀, with no change in E_max, may occur with spare receptors (Kenakin, 1993).

The smaller E_max in cells from HCT-compared to SHAM-treated animals may be due to a decrease in the number of 5-HT_1A binding sites. It has been reported that high levels of corticosterone (plasma concentrations >40 µg/dl) do not change the number of 5-HT_1A binding sites in subfield CA3 compared to sham-treated animals (Mendelson and McEwen, 1992a). However, social and restraint stress does decrease the number of 5-HT_1A binding sites in the hippocampus (Watanabe et al., 1993; McKittrick et al., 1995). One possible explanation for the discrepancy is the treatment length. Mendelson and McEwen (1992b) treated their animals with a corticosterone pellet implant for 7 days. The investigations that examined the effects of stress treated their animals for 14 days (Watanabe et al., 1993; McKittrick et al., 1995). It is possible that exposure to elevated corticosterone plasma levels must exceed 7 days before the down-regulation of 5-HT_1A binding sites occurs. Alternatively, the stress response may be activating other physiological processes, in addition to increasing corticosterone plasma levels, that induces the down regulation of 5-HT_1A binding sites.

Corticosteroids also alter the 5-HT_1A receptor-effector pathway downstream of the receptor. ADX increased although HCT decreased the magnitude of the current evoked by the activation of G proteins with GTPS. Corticosteroids could be altering G protein levels, G protein coupling or the properties of the channels linked to the G proteins. However, the attenuated 5-HT_1A mediated-response and G protein-evoked current cannot be attributed to a decrease in G protein expression. We observed that HCT treatment increased G_o and G_i1&2 -subunit levels in the hippocampus (Okuhara et al., 1997), the PTX-sensitive G proteins thought to be linked to the 5-HT_1A receptor (Andrade et al., 1986; Okuhara and Beck, 1994). Based on our results, we propose that the increased G protein levels may be a compensatory response to a decrease in effectual G protein function.

Interestingly, although corticosterone clearly had an effect on the magnitude of the outward current evoked by GTPS, there was no change in the resting membrane potential of pyramidal cells between treatment groups. This is an important observation because corticosteroid induced changes in the resting membrane potential in the presence of GTPS may not be detectable under current clamp conditions. GTPS should activate a number of different G protein-linked channels, including inward rectifying potassium channels, inducing a decrease in membrane resistance that should shunt changes in membrane potential. Alterations in the 5-HT_1A receptor-mediated response and G protein-linked current could be attributed to a change in pyramidal cell resting input resistance. However, corticosteroid treatment did not alter neuron resting input resistance in subfield CA3 pyramidal cells (Okuhara and Beck, 1998). Although we were able to detect corticosteroid-induced changes in the current evoked by the activation of G proteins we do not know if corticosterone is altering G protein function or the coupling between the G protein and potassium channel.

The potassium channel linked to the 5-HT_1A receptor signal transduction system is probably a member of the recently cloned G protein inward rectifying potassium channel family (Spauschus et al., 1996; Kobayashi et al., 1995; Lesage et al., 1994; Ponce et al., 1996; Lesage et al., 1995). ADX, ALD and HCT treatment did not alter the reversal potential, conductance or inward rectification properties of the potassium current linked to the activation of the 5-HT_1A receptor. However, alterations in potassium channel number or kinetic properties cannot be ruled out.

ADX had different effects on the 5-HT concentration response curve characteristics in cells from subfield CA1 and CA3. Previous studies reported that the concentration-response curve characteristics for the 5-HT_1A-mediated hyperpolarization in subfield CA1 were not altered by adrenalectomy or chronic activation of MR with low levels of corticosterone as compared to sham (Beck et al., 1996; Joels et al., 1991; Joels and de Kloet, 1992). In our investigation, ADX shifted the EC₅₀ value for 5-HT_1A mediated outward current compared to SHAM and ALD (chronic MR activation). The differences in these results could be explained in several ways. In this study, 1 nM corticosterone was included in the perfusion buffer of the SHAM-treated rats to maintain the basal levels of CT, whereas in the previous experiments no corticosteroid was in the perfusion buffer of the SHAM rats. We have previously demonstrated that it is necessary to maintain the treatment paradigm by including the treatment steroid in the perfusion buffer when recording from the slices on the day of the experiment (Beck et al., 1994). Previously, we reported that the 5-HT_1A receptor signal transduction system is not identical between subfields CA1 and CA3, based on different 5-HT concentration-response curve characteristics for the 5-HT_1A receptor in the two subfields (Beck et al., 1992; Okuhara and Beck, 1994). Therefore, it is possible that corticosteroids have different effects on the 5-HT_1A receptor-mediated response in subfields CA1 and CA3.

In conclusion, chronic corticosterone treatment alters the response elicited by the activation of 5-HT_1A receptors in hippocampal subfield CA3 pyramidal cells. Some of the modulatory actions of corticosterone occur downstream of the receptor, at the G protein level. Furthermore, ADX and high corticosterone treatment have opposite effects on the 5-HT_1A signal transduction system. Our results provide important information toward understanding how corticosterone modulates neurotransmitter receptor-mediated responses in the hippocampus.