Introduction

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The 5-hydroxytryptamine₇ (5-HT₇) receptor was originally identified solely by molecular biology techniques (reviewed in Eglen et al., 1997; Terrön 1998; Vanhoenacker et al., 2000). In situ hybridization and receptor binding assays in transfected cells, peripheral tissue or brain tissue revealed a distinct pharmacological profile for this receptor with a rank order affinity of 5-carboxyamidotryptamine (5-CT) > 5-HT > 5-methoxytryptamine (5-MeOT) > methiothepin > ritanserin > 8-hydroxydipropylaminotetralin (8-OH-DPAT) > clozapine > spiperone [reviewed in Eglen et al. (1997), Terrön (1998), and Vanhoenacker et al. (2000)]. The highest density of binding or in situ hybridization was located in the thalamus and CA3 subfield of the hippocampus (Ruat et al., 1993; Tsou et al., 1994; Gustafson et al., 1996; Vizuete et al., 1997; Heidmann et al., 1998). Due to the distinct pharmacological profile, regional distribution, and low sequence homology with other 5-HT receptors, this receptor was identified as the 5-HT₇ receptor.

When transfected into different cell lines, the novel 5-HT receptor was found to stimulate adenylyl cyclase activity [reviewed in Eglen et al. (1997), Terrön (1998), and Vanhoenacker et al. (2000)]. The rank order potency of agonists agreed with the rank order affinity from binding, i.e., 5-CT > 5-HT  5-MeOT > 8-OH-DPAT. Antagonists include methiothepin (pK_B = 8.1-8.5), mesulergine (pK_B = 6.7-8.0), ritanserin (pK_B = 6.4-7.4), clozapine (pK_B = 6.7-7.6), and spiperone (pK_B = 7.0-7.2). None of these antagonists are selective, however. Two selective antagonists for the 5-HT₇ receptor have been developed, i.e., SB-258719 [pK_B of 7.2 (Thomas et al., 1998)] and SB-269770 [pK_B of 8.8 (Lovell et al., 2000)].

Even though the CA3 subfield of the hippocampus contains a high density of 5-HT₇ receptor binding sites and mRNA, the physiological consequences of 5-HT₇ receptor activation are unknown. In the CA1 and CA3 subfields of the hippocampus, adenylyl cyclase activity modulates the amplitude of the sAHP that is elicited by a train of action potentials or a calcium spike (Madison and Nicoll, 1986b; Pedarzani et al., 1998). The ion channel underlying the sAHP is a calcium-activated potassium channel (reviewed in Sah, 1996). The hallmarks of the sAHP that differentiate it from other calcium-activated potassium channels include a very long time course and its modulation by the activation of neurotransmitter receptors (Sah, 1996). The sAHP amplitude is decreased by activation of -adrenergic (Madison and Nicoll, 1986a), muscarinic (Cole and Nicoll, 1984), 5-HT₄ (Torres et al., 1994), and metabotropic glutamatergic (Pedarzani and Storm, 1996) receptors. In this study we report that the sAHP amplitude is decreased by 5-HT through the activation of the 5-HT₇ receptor.

	Materials and Methods

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Procedures for preparation of hippocampal slices and intracellular electrophysiological recording are as previously described for our laboratory (Beck et al., 1992; Birnstiel and Beck, 1995). Rats (100-200 g, Harlan Sprague-Dawley) were decapitated, and the brain rapidly removed and rinsed in ice-cold artificial cerebrospinal fluid (ACSF) containing 124 mM NaCl, 3 mM KCl, 1.25 mM NaH₂PO₄, 2 mM MgSO₄, 2.5 mM CaCl₂, 10 mM dextrose, and 28 mM NaHCO₃. The hippocampus was dissected free and starting at the dorsal/septal tip sections (500-600 µm) were cut on a vibratome. Slices were placed in a holding vial containing room temperature ACSF bubbled with 95% O₂, 5% CO₂ to maintain the pH at 7.4. The slices remained in the holding vial for at least 1 h before being transferred to the recording chamber. In the recording chamber, the slice was stabilized between two nylon nets and continuously perfused with ASCF bubbled with 95% O₂, 5% CO₂ at 32 ± 1°C at a flow rate of 2 to 3 ml/min.

Standard intracellular recordings were made by pulling electrodes 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 60 to 100 M (2 M potassium methyl sulfate, 10 mM KCl). Pyramidal cells in area CA3 were impaled by briefly (10-50 ms) increasing the capacity compensation or by increasing positive current ejection through the recording electrode. The impaled neuron was hyperpolarized to facilitate sealing of the cell. Electrical signals were amplified using an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA), stored on disk for later analysis and recorded on-line on a Gould series 3200 chart recorder (Gould, Inc., Valley View, OH). Data were collected on-line with pCLAMP software (Axon Instruments). Only neurons with a resting membrane potential < 55 mV, input resistance  30 M, and action potential overshoot of at least 10 mV were used for the experiments.

The sAHP was elicited by a calcium spike. Tetrodotoxin (1 µM) and tetraethylammonium (5 mM) were included in the ACSF. A current pulse (70-100 ms) of sufficient intensity (0.8-2 nA) was used to elicit the calcium spike. Each sAHP used for data analysis was the average of three to five sAHPs elicited 30 s apart.

Drugs were added to the ACSF in known concentrations. A stock solution was made (usually 10 mM) and diluted on the day of the experiment to obtain the desired concentrations in the ACSF.

For data analysis the magnitude of the sAHP amplitude was measured during the administration of agonist and normalized by directly comparing it to the magnitude of the sAHP in the absence of agonist. The percentage inhibition for each concentration of agonist was fit to a logistic equation (E = E_max/(1 + (EC₅₀/[A])^N), where E is the response elicited by the concentration of drug (A), N is the slope, and EC₅₀ is the concentration of drug needed to elicit a response 50% of maximum. Estimates of E_max, EC₅₀, and slope were obtained for each cell and were used for statistical analyses.

Chemicals for making the stock buffer and 5-HT were obtained from Sigma Chemical Co. (St. Louis, MO). The agents 5-CT, 5-MeOT, ICS-205-930, metergoline, ritanserin, mesulergine, 1-(2,5-di-methoxy-4-iodophenyl)-2-aminopropane (DOI), clozapine, ketanserin, methiothepin, 2-methyl 5-HT, sumatriptan, and -methyl 5-HT were obtained from Research Biochemicals International (Natick, MA). WAY-100635 (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexane carboxamide) was a generous gift from Wyeth Ayerst Research (Princeton, NJ). GR-113808 ([1-[2-(methylsulfonylamino)ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate) was generously donated by Glaxo Group Research (Greenford, UK). SB-269770 ((R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol) was generously provided by SmithKline Beecham (Essex, UK).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

The magnitude of the sAHP elicited by a calcium spike in CA3 hippocampal pyramidal cells ranged from 9 to 25 mV, with a mean of 18.9 ± 0.4 mV, n = 79. The values for the sAHP amplitude were normally distributed.

To isolate any response mediated by 5-HT₇ receptor activation, other postsynaptic 5-HT receptor-mediated responses present in CA3 pyramidal cells were blocked by the addition of selective antagonists in the ACSF. Activation of a postsynaptic 5-HT_1A receptor by 5-HT leads to a very pronounced hyperpolarization of the membrane potential (Beck et al., 1992), averaging 17 mV. This hyperpolarization shunts or attenuates the magnitude of the sAHP, making it difficult to measure a 5-HT-induced inhibition of the sAHP. In previous experiments (Birnstiel and Beck 1995), spiperone, an antagonist at 5-HT_1A, 5-HT₂, and 5-HT₇ receptors, was added to the ACSF to block the 5-HT_1A hyperpolarization. However, in the presence of spiperone (10 µM), 5-HT did not inhibit the sAHP (Fig. 1); a concentration of 5-HT greater than 300 µM was needed to obtain any inhibition of the sAHP (N = 3). When the selective 5-HT_1A receptor antagonist WAY-100635 [0.1-1.0 µM, 100 and 1000 times the reported K_D at the 5-HT_1A receptor (Corradetti et al., 1996)] was added to the ACSF instead of spiperone to block the hyperpolarization, a pronounced inhibition of the sAHP amplitude by 10 µM 5-HT was seen, i.e., 60.3 ± 4.4% (N = 26) inhibition compared with the control sAHP without 5-HT. Some of the inhibition of the sAHP by 5-HT could be blocked by the selective 5-HT₄ receptor antagonist GR-113808 [0.1 and 1.0 µM, 100 and 1000 times the reported K_D for GR-113808 at the 5-HT₄ receptor (Torres et al., 1994)], i.e., 54.9 ± 5% inhibition by 10 µM 5-HT and 38.2% ± 7% inhibition by 5-HT in the presence of GR-113808 (paired t test = 2.72, P < .01, df = 14). Therefore, due to the presence of both a 5-HT_1A and 5-HT₄ postsynaptic receptor-mediated response in CA3 pyramidal cells, the selective 5-HT_1A receptor antagonist WAY-100635 (0.1-1 µM) and the selective 5-HT₄ receptor antagonist GR-113808 (0.1 µM) were included in the ACSF for all of the experiments described below.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Spiperone blocks the 5-HT inhibition of sAHP amplitude. Spiperone (10 µM) was included in the stock ACSF to block the 5-HT1A receptor-mediated stimulation of an inward rectifying potassium channel. The control sAHP is depicted in the top left corner. A concentration of 350 µM was needed to obtain any significant degree of inhibition of the sAHP amplitude. The resting membrane potential for each of the traces was 67 mV for control, 68 mV for 10 µM, 66 mV for 30 µM, 70 mV for 100 µM, and 66 mV for 350 µM.

The inhibition of the sAHP by 5-HT was concentration-dependent and reversible. Figure 2 contains sAHPs recorded from a CA3 pyramidal cell. The sAHPs were collected at the beginning of the experiment (control) and during the perfusion of the slice by the concentration of 5-HT shown above each trace. Following the administration of 5-HT, the drug was removed from the ACSF and the sAHP amplitude returned to baseline values (recovery). To save space not all of the "recovery" sAHP values are shown. In Fig. 2B, the control sAHP and the sAHP recorded in the presence of 15 µM 5-HT are superimposed. For each concentration tested, the magnitude of the sAHP recorded in the presence of 5-HT was normalized as a percentage of the sAHP collected just before each administration of 5-HT. The normalized percentage inhibition was plotted against the concentration of 5-HT and fit to a logistic equation (Fig. 2C). The form of the logistic equation is E = E_max/(1 + (EC₅₀/[A])^N), where E is equal to the response magnitude at a given concentration of ligand, A, E_max is the maximum inhibition that can be elicited by the ligand, EC₅₀ is the concentration of A that elicits a response 50% of maximum, and N is the slope.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent inhibition of the sAHP by 5-HT. The sAHP traces are the average of five sAHPs. A, following a calcium spike, the sAHP magnitude was 25 mV. WAY-100635 (0.1-1 µM) and GR-113808 (0.1 µM) were included in the buffer to block 5-HT1A and 5-HT4 receptors. In the presence of increasing concentrations of 5-HT, the magnitude of the sAHP response was reduced. In between each concentration of 5-HT the response was allowed to return to control amplitude before the next concentration of 5-HT was administered, i.e., recovery sAHPs. Not all recovery traces are shown. The resting membrane potential for each of the traces was 64 mV for control, 63 mV for 1 µM, 62 mV for 3 µM, 64 mV for recovery, 64 mV for 7 µM, 66 mV for recovery, 64 mV for 10 µM, 61 mV for 15 µM, and 66 mV for recovery. B, the control response and 15 µM response are superimposed to demonstrate that 5-HT maximally inhibited the magnitude of the sAHP. C, the magnitude of the sAHP amplitude was measured during the administration of 5-HT and normalized by directly comparing it to the magnitude of the sAHP in the absence of 5-HT. The percentage inhibition at each concentration of 5-HT was fit to a logistic equation as described under Materials and Methods.

Agonists known to have activity at the 5-HT₇ receptor in other bioassays, i.e., 5-CT and 5-MeOT, were tested and compared with 5-HT. WAY-100635 (0.1-1 µM) and GR-113808 (0.1 µM) were included in the buffer to block 5-HT_1A and 5-HT₄ receptors. Figure 3 contains sAHPs recorded from a CA3 hippocampal pyramidal cell before, during and after the administration of 5-CT. Even though recovery sAHPs were obtained following each 5-CT concentration tested, the only recovery sAHP shown is the one collected following the removal of 100 nM 5-CT from the ACSF. A similar concentration-dependent inhibition of the sAHP amplitude was obtained following 5-MeOT administration. Table 1 is a summary of the mean ± S.E. for the EC₅₀, E_max, and slope values obtained for all the cells tested with 5-HT, 5-CT, and 5-MeOT. The EC₅₀ values were significantly different between the agonists, with 5-CT demonstrating the greatest potency. The E_max and slope values were not significantly different between the three agonists.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   5-CT decreases the amplitude of the sAHP evoked by a calcium spike in a concentration-dependent manner. WAY-100635 (1.0 µM) and GR-113808 (0.1 µM) were included in the buffer to block 5-HT1A and 5-HT4 receptors. The sAHP traces are the average of five sAHPs. The concentration of 5-CT that was bath-administered is shown above each trace. In between each concentration of 5-CT the sAHP was allowed to return to baseline values, but only the recovery sAHP after the highest concentration of 5-CT is shown. The bottom right hand composite trace contains the superimposed traces obtained during control and in the presence of 100 nM 5-CT. The resting membrane potential for each trace was 72 mV for control, 72 mV for 1 nM, 72 mV for 3 nM, 72 mV for 10 nM, 70 mV for 30 nM, 68 mV for 100 nM, and 69 mV for recovery.

Figure 4 is a summary graph of the mean ± S.E. values for the inhibition of the sAHP at each of the tested concentrations of the agonists 5-HT (triangles), 5-CT (squares), and 5-MeOT (circles). The rank order potency of the agonists for the inhibition of the sAHP in the presence of WAY-100635 and GR-113808 was 5-CT > 5-HT > 5-MeOT (Fig. 4; Table 1). Other agonists known to have high or fairly selective affinity at other 5-HT receptors were tested at a concentration of 10 µM, and the magnitude of the inhibition of the sAHP by each of these ligands is presented in Table 2. None of the agonists with affinity for the 5-HT₂, 5-HT₃, 5-HT_1B, 5-HT_1D, or 5-HT₆ receptors produced any degree of inhibition of the sAHP at the concentration of 10 µM.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Summary graph containing the mean ± S.E. for each concentration of agonist tested for all of the cells tested with 5-HT (solid triangles), 5-CT (solid squares), or 5-MeOT (solid circles). WAY-100635 (0.1-10 µM) and GR-113808 (0.1 µM) were included in the buffer to block 5-HT1A and 5-HT4 receptors. The data were fitted by logistic equation, including the mean values with the error and the values that were obtained agreed with those obtained when the fitted data for each individual cell were averaged together. For 5-CT the EC50 was log 7.36, Emax = 72, and slope = 1.34; for 5-HT the EC50 was log 5.2, Emax = 58, and slope = 2.15; and for 5-MeOT the EC50 was log 5.3, Emax = 51.0, and slope = 1.4.

Antagonists known to have affinity for the 5-HT₇ receptor were tested for their ability to block the inhibition of the sAHP amplitude by 5-CT. The agonist used for these experiments was 5-CT because of its high affinity for the 5-HT₇ receptor and poor affinity for the 5-HT₄ receptor. Ritanserin, methiothepin, and mesulergine have previously been shown to be effective antagonists at the 5-HT₇ receptor [reviewed in Eglen et al. (1997), Terrön (1998), and Vanhoenacker et al. (2000)]. The concentrations of the tested antagonists were chosen to be at least 10- to 100-fold greater than the reported K_D of the antagonist for the 5-HT₇ receptor. In preliminary experiments, the equilibration time for methiothepin and mesulergine was determined to be at least 30 min. The equilibration time for the lipophilic antagonist ritanserin took over 1 h. In some cases ritanserin was added to the stock buffer at the beginning of the experiment and therefore was constantly present. WAY-100635 (1-30 µM) and GR-113808 (0.1 µM) were included in the stock buffer to block 5-HT_1A and 5-HT₄ receptors, respectively. Due to the larger concentrations of 5-CT used in the antagonist experiments, the concentration of WAY-100635 was also increased to prevent 5-CT activation of 5-HT_1A receptors.

Figure 5 contains sAHP traces taken from one cell before, during 5-CT perfusion, and during the perfusion of 5-CT in the presence of mesulergine (Fig. 5A). The graph in Fig. 5B depicts summary concentration-response information for all of the cells tested with 5-CT alone and those tested with 5-CT alone and in the presence of mesulergine. Due to the length of the experiment, instead of a complete concentration-response curve, just two concentrations of 5-CT were used for the collection of control data, i.e., 30 and 100 nM 5-CT. These data points were contiguous with those of the summated data for all of the cells tested with 5-CT alone. Mesulergine was added to the perfusion buffer, and after 30 min data for the construction of a concentration-response curve for 5-CT was collected. As can be seen, mesulergine blocked the inhibition by 5-CT and a concentration of 1 µM 5-CT was needed to obtain an inhibition of approximately 50%. Mesulergine (0.1 µM) acted as a competitive antagonist (N = 7), i.e., the E_max values were not statistically significant from 5-CT alone (Table 3). The apparent affinity (pA₂) of 7.9 was obtained using the formula pA₂ = log([concentration of antagonist]/([EC₅₀ with antagonist]/[control EC₅₀]  1)). The value used for [control EC₅₀] was the EC₅₀ from the summated 5-CT alone data, i.e., log 7.5. 

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Mesulergine blocks the inhibition of the sAHP amplitude by 5-CT. WAY-100635 (0.1-30 µM) and GR-113808 (0.1 µM) were included in the buffer to block 5-HT1A and 5-HT4 receptors. A, sAHPs recorded from a CA3 hippocampal pyramidal cell before and during the administration of 5-CT alone, and 5-CT in the presence of mesulergine (0.1 µM). Each trace is the average of five sAHPs. The concentration of 5-CT that was bath-administered is shown above each trace. The resting membrane potential for each trace was 62 mV for control, 62 mV for 30 nM, 63 mV for 100 nM, 62 mV for wash, 64 mV for 100 nM 5-CT with mesulergine, and 60 mV for 1 µM 5-CT with mesulergine. B, summary graph of all of the data for the cells tested with mesulergine. The open squares are the control responses to 5-CT (from Fig. 4), and the filled squares are the data obtained in the experiments designed to test mesulergine. The two filled square boxes that lie near the control 5-CT (open squares) concentration-response curve are the responses obtained to 30 and 100 nM 5-CT before the administration of mesulergine.

Methiothepin (0.1 µM) acted as a noncompetitive antagonist by significantly reducing the E_max of 5-CT from 72% to 48% (Fig. 6A, n = 8). The values obtained for E_max are provided in Table 3. There was a significant difference between the E_max for cells tested with methiothepin compared with 5-CT alone (Table 3).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Ritanserin is a competitive antagonist and methiothepin a noncompetitive antagonist of the 5-CT-mediated inhibition of the sAHP amplitude. WAY-100635 (0.1-30 µM) and GR-113808 (0.1 µM) were included in the buffer to block 5-HT1A and 5-HT4 receptors. In both panels the curve demarcated by open squares is the control concentration-response curve for 5-CT taken from Fig. 4. The curve on the left (filled squares) in each panel summarizes the data obtained from cells recorded in the presence of 0.1 µM methiothepin (left) and 1.0 µM ritanserin (right).

Ritanserin (1 µM) was a competitive antagonist (n = 4). Figure 6B depicts the summary data for all of the cells tested in the presence of 5-CT alone or in the presence of 5-CT with ritanserin. The calculated values of E_max, EC₅₀, and slope for 5-CT in the presence of ritanserin are summarized in Table 3. The calculated pA₂ for ritanserin was 6.8.

The selective 5-HT₇ receptor antagonist recently developed by SmithKline Beecham (Lovell et al., 2000), i.e., SB-269770, was also a competitive antagonist (Fig. 7). Figure 7A contains sAHPs recorded before and during the administration of 30 and 100 nM 5-CT and the sAHPs recorded during the administration of 5-CT in the presence of 0.1 µM SB-269770. Figure 7B contains the summary graphs for all of the cells tested with 5-CT alone or tested with 5-CT in the presence of SB-269770 (N = 3). The affinity of SB-269770 was very high, i.e., pA₂ of 8.8. 

View larger version (20K): [in this window] [in a new window]   Fig. 7.   SB-269770 is a competitive antagonist of the 5-CT-mediated inhibition of the sAHP amplitude. WAY-100635 (0.1-30 µM) and GR-113808 (0.1 µM) were included in the buffer to block 5-HT1A and 5-HT4 receptors. A, sAHPs recorded from a CA3 hippocampal pyramidal cell before and during the administration of 5-CT alone, and 5-CT in the presence of SB-269770 (0.1 µM; SB). Each sAHP is the average of five sAHPs. The resting membrane potential for each trace was 62 mV for control, 61 mV for 30 nM, 61 mV for 100 nM, 61 mV for wash with SB, 61 mV for 300 nM with SB, 61 mV for 1 µM with SB, 62 mV for 3 µM with SB, and 62 mV for 10 µM with SB. B, summary graph of all of the data for the cells tested with SB-269770 (0.1 µM). The open squares are the control responses to 5-CT (from Fig. 4) and the filled squares are the data obtained in the experiments designed to test SB-269770.

Other antagonists known to have affinity at other 5-HT receptors were tested for their ability to block the inhibitory effect of 5-CT on the calcium-induced sAHP. In the presence of the 5-HT₂/5-HT₆/5-HT₇ receptor antagonist clozapine (10 µM) or the 5-HT₃ antagonist ICS-205,930 (100 nM) no differences were found in the percentage inhibition of the sAHP amplitude by 100 nM 5-CT, i.e., 52 ± 7% inhibition in control (N = 5), 43 ± 16 with clozapine (N = 3), and 45 ± 5% inhibition with ICS-205,930 (N = 2). Unexpectedly, clozapine did not have a large effect as an agonist or antagonist. As an antagonist at a concentration of 10 µM, it reduced the maximal inhibition by only 30%.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Using intracellular recording techniques in area CA3 of a hippocampal slice preparation maintained in vitro, 5-HT reduced the amplitude of the sAHP in area CA3 hippocampal pyramidal cells in a reversible, concentration-dependent manner. The inhibition of the sAHP amplitude was through activation of the 5-HT₇ receptor.

The mainstay of pharmacology for the characterization of a neurotransmitter receptor is to compare the rank order potency of agonists and absolute affinity of antagonists. The 5-HT₇ receptor was originally identified solely by molecular biology techniques. Affinity measurements using receptor binding assays in transfected cells, peripheral tissue, or brain tissue revealed a distinct pharmacological profile for this receptor with a rank order affinity of 5-CT > 5-HT = 5-MeOT > methiothepin > ritanserin > 8-OH-DPAT > clozapine > spiperone [reviewed in Eglen et al. (1997), Terrön (1998), and Vanhoenacker et al. (2000)]. The highest density of binding or in situ hybridization was located in the thalamus and CA3 subfield of the hippocampus (Ruat et al., 1993; Tsou et al., 1994; Gustafson et al., 1996; Vizuete et al., 1997; Heidmann et al., 1998).

Various bioassays have been used to obtain rank order affinity of agonists and antagonists for the 5-HT₇ receptor. Activation of the 5-HT₇ receptor in transfected cell lines leads to stimulation of adenylyl cyclase [reviewed in Eglen et al. (1997), Terrön (1998), and Vanhoenacker et al. (2000)]. In the periphery 5-HT₇ receptor activation mediates constriction or dilation of vasculature smooth muscle, hypotension, tachycardia, and phase shift in circadian rhythms [reviewed in Eglen et al. (1997), Terrön (1998), and Vanhoenacker et al. (2000)]. The rank order potency of agonists in these bioassays agreed with the rank order affinity from binding, i.e., 5-CT > 5-HT = 5 MeOT > 8-OH-DPAT. The rank order potency of the competitive antagonists at the 5-HT₇ receptor is methiothepin (pK_B = 8.1-8.5) > mesulergine (pK_B = 6.7-8.0) > ritanserin (pK_B = 6.4-7.4) > clozapine (pK_B = 6.7-7.6) > spiperone (pK_B = 7.0-7.2).

In this study, the rank order potency of the tested agonists for the inhibition of the sAHP amplitude in CA3 hippocampal pyramidal cells was 5-CT > 5-HT  5-MeOT. Agonists known to have affinity at other 5-HT receptors, 5-HT₂, 5-HT_1B, 5-HT_1D, 5-HT₃, and 5-HT₆, did not have any measurable effect on the sAHP. This rank order of agonist affinity is seen for both the 5-HT₇ and 5-HT_1A receptors (Terrön, 1998). However, in the experiments described here the 5-HT_1A receptor was blocked by the selective antagonist WAY-100635 (Corradetti et al., 1996). This rank order potency of agonists agrees with that reported for other bioassay systems for 5-HT₇ receptor activation as discussed above.

The agonist with the highest potency for inhibiting the sAHP was 5-CT. This agonist has nanomolar affinity for 5-HT_1A and 5-HT₇ receptors with much less affinity (10- to 100-fold) for the 5-HT_1B and 5-HT_1D receptors; affinity for other 5-HT receptors is negligible. Therefore, 5-CT is currently the most selective agonist available for the characterization of the 5-HT₇ receptor and was the agonist used in the experiments to determine the ability of antagonists to block 5-HT₇ receptor-mediated inhibition of the sAHP.

Receptor characterization is dependent on rank order affinities of antagonists and their absolute affinity. Initially, spiperone (10 µM) was used as an antagonist to block the 5-HT_1A-mediated hyperpolarization. However, in the presence of spiperone, 5-HT did not reduce the sAHP amplitude. Spiperone has affinity for both the 5-HT_1A as well as the 5-HT₇ receptor [reviewed in Eglen et al. (1997) and Terrön (1998)]. When the selective 5-HT_1A receptor antagonist WAY-100635 was used at a concentration that was 100 to 1000 times its reported K_D to block 5-HT_1A receptor activation instead of spiperone, the inhibition of the sAHP by 5-HT was apparent.

Ritanserin and mesulergine acted as competitive antagonists to the inhibition of the sAHP by 5-CT with apparent pA₂ values of 6.8 and 7.9, respectively. An apparent affinity value could not be obtained for methiothepin, because it was a noncompetitive antagonist. In previous studies using bioassays to measure 5-HT₇ receptor-mediated actions, methiothepin has been reported to be an insurmountable antagonist (McLean and Coupar, 1996; Terron, 1997; Terron and Falcon-Neri, 1999). The selective 5-HT₇ receptor antagonist SB-269770 was the most effective antagonist with a pA₂ value of 8.8. The rank order potency, i.e., SB-269770 > mesulergine > ritanserin, and the absolute affinity values we obtained for the antagonists effective at blocking the 5-CT inhibition of the sAHP amplitude agree well with previous reports for these antagonists acting at the 5-HT₇ receptor (Thomas et al., 1998) [reviewed in Eglen et al. (1997), Terrön (1998), and Vanhoenacker et al. (2000)].

One surprising result was the small effect of clozapine as an agonist or antagonist. At a concentration of 10 µM it was ineffective at producing any significant block of the 5-CT-mediated inhibition of the sAHP. Although many studies report that clozapine is an effective competitive antagonist (Leung et al., 1996; Hirst et al., 1997; Kitazawa et al., 1998), other studies have found that clozapine is relatively inactive at 5-HT₇ receptors either as an antagonist, i.e., with micromolar affinity (De Vries et al., 1997; Villalon et al., 1997), or as a partial agonist (Terron, 1996; Villalon et al., 1997).

Very little is known regarding the biological significance of 5-HT₇ receptor activation in the central nervous system. An increasing body of evidence is becoming available to support the theory that the 5-HT₇ receptor plays a role in psychiatric disease states. The regional distribution of the 5-HT₇ receptor includes limbic areas, and the binding profile includes high affinity to antidepressants, antipsychotics, and hallucinogens [reviewed in Eglen et al. (1997), Terrön (1998), and Vanhoenacker et al. (2000)]. Regulation of the 5-HT₇ receptor-effector pathway may underly the clinical efficacy of certain classes of antidepressant drugs and/or the etiology of neuropsychiatric disease states such as depression. Chronic treatment with the serotonin selective reuptake inhibitor fluoxetine decreases the 5-HT₇ receptor number in hypothalamus (Sleight et al., 1995; Mullins et al., 1999) and enhances 5-HT₇ receptor-mediated stimulation of adenylyl cyclase activity in frontal cortical astrocytes (Shimizu et al., 1996). Removal of the stress hormone corticosterone by adrenalectomy or treatment with the synthetic stress hormone dexamethasone increases and decreases the 5-HT₇ receptor number, respectively (Le Corre et al., 1997; Shimizu et al., 1997).

A high density of the 5-HT₇ receptor within the CA3 subfield of the hippocampus positions this receptor to be of primary importance in the regulation of the neural circuitry underlying brain wave activity. The 5-HT-hippocampal-CA3 circuit is a primary regulator of electroencephalogram activity and sleep patterns (Vertes et al., 1994; Kinney et al., 1995; Maru et al., 1979; Varga et al., 1998), and disrupted circadian rhythms and/or sleep patterns are often seen in individuals with psychiatric disorders (reviewed in Thase, 1998). One of the primary factors that regulates the frequency of theta brain wave activity that originates in the CA3 subfield of the hippocampus is the sAHP. Increasing the amplitude of the sAHP decreases theta frequency, whereas decreasing the amplitude of the sAHP increases theta frequency (Traub et al., 1992). Our findings that 5-HT₇ receptor activation decreases the sAHP amplitude provides an important link in understanding how the 5-HT-hippocampal-CA3 circuit regulates theta activity.

Our results provide important new information on the physiological function of the 5-HT₇ receptor. Based on the agreement of the rank order potencies of agonists and antagonists, and the pA₂ values for the antagonists with the known values for the 5-HT₇ receptor in other bioassay systems, the conclusion is that one of the receptors mediating the decrease in sAHP amplitude in CA3 hippocampal pyramidal cells is the 5-HT₇ receptor. The 5-HT₇ receptor-mediated inhibition of the sAHP amplitude in CA3 hippocampal pyramidal cells should be an important bioassay for use in the development of new compounds that are selective for the 5-HT₇ receptor. The identification of the 5-HT₇ receptor-mediated response allows for further investigations into the regulation of this receptor by drugs used in the treatment of psychoactive disease states and by chronic stress and into how this receptor may regulate hippocampal function.