Introduction

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The 5-HT neurotransmitter system (Gjerris et al., 1987; Stokes, 1993; Meltzer and Lowy, 1987; Blier et al., 1987; Robinson, 1993; Cowen, 1993) and the raphe-hippocampal pathway have been implicated in the etiology and treatment of complex emotional disorders such as depression (Wong et al., 1995; Bremner et al., 1995; Axelson et al., 1993). The raphe-hippocampal 5-HT system is a likely target for psychoactive drugs used to treat such disease states. Clinical efficacy of antidepressants is usually not apparent for at least 2 to 3 weeks after the start of treatment. Even though the acute effects of antidepressant agents have been determined, i.e., 5-HT uptake inhibition, norepinephrine uptake inhibition and MAO inhibition, the long-term mechanism of action of antidepressant drugs at the cellular level has not been clearly defined.

Fluoxetine is a SSRI and is one of the main drugs used for the treatment of depression (Wong et al., 1995; Leonard, 1993; Stokes, 1993). Previous studies have investigated the effects of chronic fluoxetine treatment on components of the 5-HT-receptor-effector pathway. The 5-HT₁ and 5-HT₂ receptor systems have been studied the most. Several laboratories have reported that chronic fluoxetine treatment does not alter the number of 5-HT_1A or 5-HT₂ binding sites in the hippocampus as assessed by homogenate assays (Klimek et al., 1994; Welner et al., 1989; Goodnough and Baker, 1994); others have reported an increase in 5-HT₂ sites in the CA2-3 subfield of the hippocampus (Hrdina and Vu, 1993; Klimek et al., 1994). Chronic fluoxetine treatment has been shown to have no effect on (Varrault et al., 1991), or to decrease, 5-HT_1A receptor-mediated inhibition of adenylyl cyclase activity in hippocampus (Newman et al., 1992). G protein  subunit levels are not altered in the hippocampus by chronic fluoxetine treatment (Lesch et al., 1992).

Previous electrophysiological studies have measured the effects of chronic antidepressant drug treatment, including tricyclic antidepressants, MAO inhibitors and SSRIs such as fluoxetine, by measuring changes in extracellularly recorded dorsal raphe and CA3 hippocampal pyramidal cell firing rate elicited by 5-HT (for review, see Chaput et al., 1991; Blier et al., 1987). These studies have implicated an increased sensitivity of the 5-HT neurotransmitter system. Specifically, it has been suggested that fluoxetine decreases the release of 5-HT from presynaptic terminals in CA3 after chronic fluoxetine treatment; no change was found in the postsynaptic 5-HT-elicited responses in either the dorsal raphe or the CA3 subfield of the hippocampus (Blier et al., 1988).

Intracellular recording techniques are useful for determining whether chronic drug treatment alters basic cell characteristics and for determining the mechanism of action underlying neurotransmitter-elicited changes recorded by extracellular recording techniques. Only one intracellular recording study has been conducted to determine the long-term modulatory action of the tricyclic antidepressant imipramine on 5-HT receptor function in area CA1 of the hippocampus (Beck and Halloran, 1989). The long-term effects of chronic fluoxetine treatment on the physiological response of hippocampal pyramidal cells has not been determined using intracellular recording techniques.

The passive and active cell characteristics of CA1 and CA3 hippocampal pyramidal cells are different (Beck et al., 1992; Brown et al., 1981; Spruston and Johnston, 1992), i.e., the input resistance and time constant of the CA3 cells are greater than those of the CA1 cells. This distinction can be attributed to differences in the types, densities and distribution of ion channels between the CA1 and CA3 pyramidal neurons. Also, the concentration-response curves characteristics of the 5-HT_1A and GABA_B receptor-mediated responses are different in CA3 than in CA1. These differences are attributed to differences in receptor-effector number, receptor-effector coupling or recognition site of the receptors (Beck et al., 1992; Beck et al., 1995). Our hypothesis is that the effects of chronic treatment with fluoxetine on hippocampal pyramidal cell characteristics and/or on receptor-mediated responses will not be the same in the CA1 and CA3 subfields of the hippocampus because the ion channels and receptor-effector coupling are not the same in the two subfields. The purpose of the experiments reported here was to determine the modulatory effects of chronic fluoxetine treatment on pyramidal cell characteristics and on 5-HT receptor-mediated responses in areas CA1 and CA3 of the hippocampus. Because 5-HT and GABA receptor systems have been found to share receptor-effector components (Andrade et al., 1986; Okuhara and Beck, 1994), GABA_B receptor-elicited responses were measured for comparison.

	Materials and Methods

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Antidepressant treatment. Male Sprague-Dawley rats (75-125 g) were anesthetized with ether, and an osmotic minipump (Alzet, Pala Alto, CA) implanted s.c. in the back. Fluoxetine hydrochloride was dissolved in 50% DMSO at a concentration of 11.7 g/l to produce an average dose of 6.68 mg/kg at the final weight of approximately 250 g for the rat after 3 weeks of treatment. Therefore, at the beginning of the treatment, the dose of fluoxetine would be higher because the rat weighed less. The control group of rats were implanted with minipumps containing 50% DMSO or physiological saline.

Hippocampal slice preparation. The rats were anesthetized with ether and decapitated. Trunk blood was collected in centrifuge tubes containing 0.3 ml of a solution of 0.3 M EDTA (pH = 7.4) and 1000 KIU of trasolol. The blood was centrifuged at 8600 rpm for 20 min at 4°C, and the plasma was removed and frozen. Plasma fluoxetine and norfluoxetine levels were determined by a solid-phase extraction procedure developed by the Toxicology/Psychopharmacology laboratory at the Hines VA hospital. Separation, identification and quantification of the two drugs were accomplished by HPLC. The brain was rapidly removed and rinsed in ice-cold ACSF containing (mM): NaCl (124), KCl (3), NaH₂PO₄ (1.25), MgSO₄ (2), CaCl₂ (2.5), dextrose (10), NaHCO₃ (28). The hippocampus was dissected free, and, starting at the dorsal/septal tip, 500 to 600-µ sections were cut on a vibratome. Slices were placed in a holding vial containing room-temperature ACSF bubbled with 95% O₂/5% CO₂, pH = 7.4. After at least 1 h, a slice was transferred to the recording chamber, where it was perfused continuously with ACSF at 32°C ± 1°C and bubbled with 95% O₂/5% CO₂ at a flow rate of 2 to 3 ml/min. Fluoxetine was not included in the ACSF.

Intracellular recording techniques. Intracellular recordings were made as previously described (Beck et al., 1992). Electrodes were pulled from borosilicate capillary tubing (1.2 mm O.D., 0.69 mm I.D., Sutter Instruments, Novato, CA) on a Brown and Flaming electrode puller (Sutter Instruments, Novato, CA) to obtain resistances of 40 to 140 M (2 M KCl or 2 M KCH₃SO₄/10 mM KCl). Pyramidal cells in area CA1 or CA3 were impaled by briefly (10-50 ms) increasing the capacitance compensation or by briefly increasing positive current ejection through the recording electrode. The pyramidal cells were hyperpolarized after impalement to facilitate sealing, which usually took from 15 to 30 min. Electrical signals were amplified using an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA), stored on disk for later analysis using pClamp software (Axon Instruments, Foster City, CA) and recorded on a Gould chart recorder, Series 2200 or 3200 (Gould Incorporated, Cleveland, OH).

Concentration-response curves. Drugs were tested by the addition of concentrated stock drug solution into a reservoir of 50 to 60 ml of ACSF. For concentration-response curves, four to seven concentrations of drug were tested in increments of at least one-half log unit.

Statistical analyses. Our hypothesis was that fluoxetine would alter the cell characteristics, and/or the characteristics of the 5-HT or baclofen concentration-response curves for 5-HT_1A, GABA_B and/or 5-HT₄ receptor activation. Previously, we and others have demonstrated that there were significant differences between the two hippocampal subfields in cell characteristics (Beck et al., 1994; Brown et al., 1981; Spruston and Johnston, 1992) and concentration-response characteristics for 5-HT (Beck et al., 1992) and baclofen (Beck et al., 1995). To confirm these previous findings, an ANOVA was conducted comparing the control data from CA1 with the control data from CA3. To test for modulatory actions of fluoxetine, an ANOVA was conducted on data within a particular subfield. Data are reported as mean ± S.E.M. The geometric means of the EC₅₀ values were used for statistical comparisons. A P < .05 was considered significant.

Chemicals and drugs. The chemicals for making the ACSF and 5-HT hydrochloride were purchased from Sigma (St. Louis, MO). Fluoxetine was generously donated by Eli Lilly (Indianapolis, IN). Baclofen was generously donated by Ciba-Geigy (Suffern, NY). DMSO was obtained from Fisher Scientific (Pittsburgh, PA).

	Results

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Data were obtained from 12 rats treated with fluoxetine and 16 sham control rats; 68 total cells, 34 from fluoxetine treated rats and 34 from sham-treated control rats. Each individual cell was treated as an independent sample, even though more than one cell may have been obtained from each rat.

Fluoxetine plasma levels. On the day of the experiment, trunk blood was collected, and plasma fluoxetine and norfluoxetine levels were determined. The data recorded from cells from rats that had detectable plasma fluoxetine and norfluoxetine levels were used for analysis as data from treated rats; data from two rats with no detectable plasma levels of norfluoxetine or fluoxetine were discarded. Control animals had fluoxetine levels and norfluoxetine levels that were not detectable (n = 3). For the treated rats, the fluoxetine levels were 107 ± 15 ng/ml (range 65-200) and norfluoxetine levels were 118 ± 22 ng/ml (range 19-200 ng/ml), n = 12.

Cell properties. The passive and active characteristics of the CA1 and CA3 hippocampal pyramidal cells from control and fluoxetine-treated rats are summarized in table 1. Previous studies have reported that the passive cell characteristics are not the same in CA1 and CA3 pyramidal cells (Beck et al., 1994; Brown et al., 1981; Spruston and Johnston, 1992), i.e., the input resistance and the time constant of the CA3 cells are greater than those of the CA1 cells. The results of the experiments reported here confirmed those studies, i.e., the input resistance measured at 300 ms and the time constant of the CA3 cells were significantly greater than those of the CA1 cells (table 1).

5-HT_1A receptor-mediated hyperpolarization. As previously reported, 5-HT elicited a hyperpolarization in both CA1 and CA3 hippocampal pyramidal cells (fig. 1). Also in confirmation of previous findings (Beck et al., 1992), the magnitude of the hyperpolarization was greater in area CA3 than in area CA1, and 5-HT was more potent in area CA1 than in area CA3 (table 2; fig. 2).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   A) Individual chart recording of 5-HT1A receptor-mediated hyperpolarization in a CA1 hippocampal pyramidal cell from a fluoxetine-treated rat. The length of the line above the chart recording indicates the amount of time that 5-HT was in the ACSF. The concentration of 5-HT is given on the top of the line. Downward deflections are membrane potential responses to 300 pA hyperpolarizing current pulses to monitor membrane resistance. Once the response had reached steady state, five 900-pA, 300-ms current pulses were applied to generate a train of action potentials for the measurement of the sAHP. If necessary, direct current was applied intracellularly to return the membrane potential to the resting membrane potential level before the sAHPs were elicited in the presence of 5-HT. Resting membrane potential was 65 mV. B) sAHP traces taken at a faster sampling rate generated in response to 900-pA, 300-ms current pulse before (Control) and during (5-HT 30 µM) 5-HT administration. Each sAHP trace is the average of five individual traces. Data in panels A and B are from the same cell.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Summary graphs of 5-HT concentration-response curves for the 5-HT1A receptor-mediated hyperpolarization in areas CA1 and CA3 in control and fluoxetine-treated cells. Note that the y axis is different for the CA1 and CA3 summary graphs. The number of neurons per data point was 9 to 15 for CA1 and 6 to 9 for CA3. Values are mean ± S.E.M.

GABA_B receptor-mediated hyperpolarization. Baclofen elicits a hyperpolarization in both area CA1 and area CA3, and the GABA_B receptor and the 5-HT_1A receptor appear to share some component of the receptor-effector pathway (Okuhara and Beck, 1994; Andrade et al., 1986). The data from the CA1 and CA3 cells from the sham-treated rats were reported in a previous study (Beck et al., 1995), which describes the finding that baclofen is less potent at eliciting a hyperpolarization through activation of the GABA_B receptor in area CA1 than in CA3 (table 3). Like the 5-HT-elicited hyperpolarization, the characteristics of the baclofen concentration-response curve for the GABA_B receptor-mediated hyperpolarization differ in area CA1 and area CA3 hippocampal pyramidal cells (Beck et al., 1995). However, unlike the 5-HT_1A response, the GABA_B response is less potent in CA1 than in CA3. Like the 5-HT_1A response, the maximal response elicited by baclofen is greater in CA3 than in CA1 (Beck et al., 1995).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Summary graphs of baclofen concentration-response curves for the GABAB receptor-mediated hyperpolarization in areas CA1 and CA3 in control and fluoxetine treated cells. Note that the y-axis scale is different for the CA1 and CA3 summary graphs. The number of neurons per data point for CA1 was 6 to 10, and 3 to 6 neurons for CA3. Values are mean ± S.E.M.

5-HT₄ receptor-mediated decrease in sAHP amplitude in area CA1. Five sAHPs were generated by a 900-pA, 300-ms current pulse both before and during the perfusion of the slice with 5-HT. The five sAHPs were averaged, and the amplitude and the half-decay time of the sAHP were measured. During the hyperpolarization elicited by 5-HT, the resting membrane potential was brought back to base-line levels by injecting direct current so that the sAHP could be generated (fig. 1). In the CA3 subfield, the magnitude of the hyperpolarization was very large; because of the large increase in potassium conductance, even with direct current injection to bring the membrane potential back to base-line levels, it was not possible to generate a train of action potentials to elicit the sAHP in every cell. Therefore, data on the chronic effects of fluoxetine treatment on the 5-HT-elicited decrease in sAHP amplitude were not obtained from area CA3 pyramidal cells.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Summary graphs of 5-HT concentration-response curves for the 5-HT4 receptor-mediated decrease in sAHP amplitude (panel A) and half-decay time (panel B). The values are given as percent decrease compared with control. The number of neurons per data point was 7 to 13. Values are mean ± S.E.M.

	Discussion

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The major findings of this study were that chronic fluoxetine treatment selectively altered the cell characteristics and the concentration-response curve characteristics of 5-HT and baclofen to elicit a membrane hyperpolarization in area CA1 hippocampal pyramidal cells. In contrast, chronic treatment with fluoxetine did not alter the pyramidal cell characteristics or the concentration-response curve characteristics in area CA3 hippocampal pyramidal cells.

The differences in the passive cell characteristics between CA1 and CA3 pyramidal cells, as previously noted (Brown et al., 1981; Bilkey and Schwartzkroin, 1990; Beck et al., 1994), were confirmed by the results collected in this experiment. Interestingly, the chronic effects of fluoxetine were manifested only on the cell characteristics of the CA1 neurons. The "sag" in membrane potential, measured by the difference in resistance measured at approximately 100 ms and 300 ms, was greater in fluoxetine-treated animals. This "sag" is thought to be due to the activation of a mixed cationic conductance termed I_Q or I_h (Brown et al., 1990; Halliwell and Adams, 1982). Further experiments will be necessary to determine which ionic conductance(s) were changed to account for the measured increase in resistance at 300 ms. Chronic fluoxetine treatment also increased the time constant and the half-decay time of the sAHP of CA1 pyramidal cells. A change in the time constant could be due to an alteration in input resistance and/or capacitance and could account for the increased "sag" recorded in fluoxetine-treated cells. The increased time constant would make the cell more sensitive to synaptic activity and increase the likelihood of synaptic potentials summating. In contrast, the prolonged time course of the sAHP would decrease the excitability of the cell and decrease the probability that synaptic input would summate. Therefore, the net effect of chronic fluoxetine would make the cell more responsive at low synaptic activity and less responsive to large, prolonged synaptic input.

Chronic fluoxetine treatment altered the EC₅₀ for both the 5-HT and baclofen concentration-response curves for the 5-HT_1A and GABA_B receptor-mediated hyperpolarization of area CA1 pyramidal cells, but not area CA3 pyramidal cells. Fluoxetine was present in the plasma and brain on the day of the experiment, because the fluoxetine minipumps were not removed before preparation of the brain slices. The acute effect of fluoxetine is to prevent the uptake of 5-HT into nerve terminals; one explanation for the increase in potency of the 5-HT_1A receptor-mediated hyperpolarization in area CA1 is that it is due to an acute effect of fluoxetine. Previous studies have reported on the acute effects of fluoxetine added to the perfusion buffer of hippocampal brain slices maintained in vitro. In area CA1, fluoxetine administration led to a shift to the left in the threshold concentration for the 5-HT concentration-response curve for eliciting a 5-HT_1A receptor-mediated hyperpolarization, but the EC₅₀ was not altered (Andrade and Nicoll, 1987). In area CA3, fluoxetine did not alter the EC₅₀ of the 5-HT concentration-response curve (Beck et al., 1992). In this study, there was a significant shift to the left in the EC₅₀ and a decrease in the slope of the 5-HT concentration-response curves recorded in cells from rats treated with fluoxetine for 3 weeks. Therefore, because the acute effects of fluoxetine did not result in significant changes in the EC₅₀ or E_max of the concentration-response curves for the 5-HT_1A receptor-mediated hyperpolarization, our results cannot be attributed to an acute effect of residual fluoxetine. Also, the amount of time before a cell is obtained after the preparation of the tissue ranges from 2 h to 10 h. Any residual fluoxetine present in the tissue after decapitation is probably removed in that amount of time. Therefore, we conclude that the changes in EC₅₀ and slope of the 5-HT and baclofen concentration-response curves are due to the chronic effects of fluoxetine.

The alteration of the 5-HT and baclofen concentration-response curves by chronic fluoxetine was in the opposite direction, i.e., an increase in potency for the 5-HT_1A receptor-mediated response and a decrease in potency of the GABA_B response. The GABA_B and 5-HT_1A receptors share some component(s) of their effector pathways, because no additivity is measured when saturating concentrations of 5-HT and baclofen are administered together (Andrade et al., 1986; Okuhara and Beck, 1994). If fluoxetine was modulating some shared component of the pathway, the changes in the concentration-response curve characteristics for 5-HT and baclofen would be expected to be similar. Chronic fluoxetine treatment could be altering any component of the receptor-effector pathway: receptor number, coupling efficiency between receptor and G protein or G protein and ion channel, number of G proteins, number of ion channels or kinetics of the ion channel. However, because we found that fluoxetine treatment produced differential effects on the 5-HT_1A and GABA_B receptor-mediated hyperpolarization, we propose that fluoxetine is altering some component of the receptor-effector pathway that is not shared by these two receptor-effector pathways.

Differences in receptor number cannot account for the divergence in the E_max values for 5-HT_1A or GABA_B receptor-mediated responses recorded in area CA1 and CA3 in sham-treated rats (Beck et al., 1992; Beck et al., 1995). The density of 5-HT_1A and GABA_B receptors is higher in area CA1 than in area CA3 of the hippocampus (Chu et al., 1990; Knott et al., 1993; Pazos and Palacios, 1985; Ahlenius and Larsson, 1987; Welner et al., 1989; Gozlan et al., 1995). The density of the GABA_B receptors is relatively homogenous in the cell body and dendritic fields of both areas CA1 and CA3 (Chu et al., 1990; Knott et al., 1993). Within the CA1 subfield, the 5-HT_1A receptors are in all of the layers; in area CA3 the receptors are located in stratum oriens. The larger input resistance of the CA3 cells can only partially account for the larger magnitude of the response elicited by 5-HT_1A and GABA_B receptor activation in area CA3 as compared with area CA1; the mechanism underlying this difference has not been definitively identified.

Previous studies have shown that fluoxetine treatment did not alter the amount of binding or the affinity of 5-HT or of the 5-HT_1A agonist 8-hydroxy-2-dipropylamino-tetralin using hippocampal homogenate binding assays (Maggi et al., 1980; Peroutka and Snyder, 1980) or in area CA1 or area CA3 using autoradiography techniques (Klimek et al., 1994; Welner et al., 1989). Therefore, because there is no change in the affinity or density of the 5-HT_1A receptors, the shift in the slope and potency of the 5-HT concentration-response curve for the 5-HT_1A receptor-mediated hyperpolarization in area CA1 cannot be attributed to changes in receptor number or affinity. To our knowledge, no one has assessed GABA_B receptor density in the hippocampus after chronic fluoxetine treatment. One study reported an up-regulation of GABA_B receptors in the frontal cortex after chronic fluoxetine treatment (Lloyd et al., 1985).

The identity of the G protein(s) that link the 5-HT_1A and GABA_B receptors in areas CA1 and CA3 to the potassium channel have not been identified, though it is known that they are pertussis toxin-sensitive (Andrade et al., 1986; Okuhara and Beck, 1994). It is entirely possible that the 5-HT_1A and GABA_B receptors are not linked to the same G protein in either or both hippocampal subfields. We have found that the distribution of the pertussis toxin-sensitive G proteins G_i and G_o is different in areas CA1 and CA3 (Okuhara et al., 1996). In area CA1, G_o labeling was found in the apical and distal dendrites, whereas it was very diffuse in the neuropil of area CA3 neurons. In area CA3, G_i labeling was fibrous and was concentrated in patches surrounding the pyramidal cell, but it was primarily in the neuropil in the area CA1 neurons (Okuhara et al., 1996). Fluoxetine treatment had no effect on the density of G protein mRNA in whole hippocampus (Lesch et al., 1992). It is possible that chronic fluoxetine treatment has selective effect(s) on G protein number or distribution within the subfields of the hippocampus.

Previous studies have demonstrated that the 5-HT_1A receptor also inhibits forskolin-stimulated adenylyl cyclase activity through a pertussis toxin-sensitive G protein (De Vivo and Maayani, 1986). Chronic fluoxetine treatment has been reported to decrease the 5-HT_1A receptor-mediated inhibition of adenylyl cyclase (Newman et al., 1992) or to have no effect (Varrault et al., 1991) in whole hippocampal homogenates. Because the 5-HT_1A receptor-mediated hyperpolarization is not mediated through a change in adenylyl cyclase activity (Andrade et al., 1986) the effects of fluoxetine cannot be attributed to a modification of adenylyl cyclase activity.

The 5-HT₄ receptor-mediated decrease in sAHP amplitude recorded in area CA1 was not altered by fluoxetine treatment. This receptor-mediated response was not recorded in isolation, but concomitantly with the 5-HT_1A receptor-mediated hyperpolarization. Approximately a 15% decrease in sAHP amplitude occurs as a consequence of the opening of the inward rectifying potassium channel after 5-HT_1A receptor activation (Andrade and Nicoll, 1987). The shift to the left in the concentration-response curve for 5-HT for the decrease in sAHP amplitude could be due primarily to the larger magnitude of the hyperpolarization elicited by lower 5-HT concentrations in fluoxetine-treated animals. It is interesting that fluoxetine selectively altered the 5-HT_1A receptor-mediated response and did not alter the 5-HT₄ response. The effects of fluoxetine cannot be ascribed to a general nonspecific action on all 5-HT receptors.

In conclusion, chronic fluoxetine treatment had selective actions on neurotransmitter receptor-mediated responses across hippocampal subfields and within a subfield. Fluoxetine selectively modulated area CA1 hippocampal pyramidal cell characteristics, as well as 5-HT_1A and GABA_B receptor-mediated effects in area CA1, without altering area CA3 hippocampal pyramidal cell characteristics or neurotransmitter actions. Also, fluoxetine selectively altered 5-HT_1A, but not 5-HT₄ receptor-mediated responses within area CA1. The actions of fluoxetine on the 5-HT_1A and GABA_B receptor-mediated hyperpolarization were in the opposite direction, even though it has been shown that these neurotransmitter receptors share some component(s) of their receptor-effector pathway. We conclude that the effects of fluoxetine are not diffuse but selective and that they appear to be on some component(s) of the receptor-effector pathways that are not shared across neurotransmitter systems or hippocampal subfields.