Introduction

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The effects of serotonin (5-hydroxytryptamine, 5-HT) in the central nervous system are mediated by multiple serotonin receptors. There are currently 14 receptor subtypes known, classified into seven receptor subfamilies based on their sequence homology, pharmacology, and signal transduction cascade (Hoyer et al., 1994). During the last two decades, there has been a growing understanding of the electrophysiological response mediated by many of these different receptor subtypes (Andrade, 1998; Barnes and Sharp, 1999). However, this understanding is still incomplete. There are receptor subtypes that have no identified electrophysiological function, and there are also well characterized physiological responses to 5-HT to which no known receptor can be assigned. Outstanding among the first of these is the 5-HT₇ receptor subtype, a subtype that is well characterized from a pharmacological standpoint, but poorly characterized in terms of in situ electrophysiology in the brain.

The 5-HT₇ receptor was originally cloned from several mammalian species, including rat (Lovenberg et al., 1993; Meyerhof et al., 1993; Ruat et al., 1993; Shen et al., 1993), mouse (Plassat et al., 1993), guinea pig (Tsou et al., 1994), and human (Bard et al., 1993), based on its homology to other serotonin receptors. As determined from sequence analysis, this serotonin receptor is part of the G-protein superfamily of receptors containing the typical seven transmembrane regions (Lovenberg et al., 1993; Meyerhof et al., 1993; Plassat et al., 1993; Shen et al., 1993; Tsou et al., 1994; Stam et al., 1997). In heterologous expression systems, the 5-HT₇ receptor increases cAMP accumulation when stimulated by 5-HT (Meyerhof et al., 1993; Plassat et al., 1993; Shen et al., 1993; Tsou et al., 1994; Stam et al., 1997) and hence is thought to couple to G_s.

The 5-HT₇ receptor pharmacology has been extensively studied in heterologous expression systems. In such systems, the 5-HT₇ receptor can be stimulated with 5-HT as well as 5-HT₁ agonists. For example, the rank order of potency at this receptor for several commonly used serotonin agonists is 5-carboxamidotryptamine (5-CT) > 5-HT  5-methoxytryptamine (5-MeOT) > 8-hydroxydipropylaminotetralin (8-OHDPAT) 2,5-dimethoxy-4-iodoamphetamine (DOI) (Lovenberg et al., 1993; Plassat et al., 1993; Ruat et al., 1993; Hoyer et al., 1994; Adham et al., 1998). Conversely, many compounds traditionally considered 5-HT₂ antagonists, such as ritanserin, methysergide, or mesulergine, are effective 5-HT₇ antagonists (Bard et al., 1993; Lovenberg et al., 1993; Shen et al., 1993). Thus, the 5-HT₇ receptor can be characterized broadly as a receptor activated by "5-HT₁ " agonists, but inhibited by "5-HT₂ " antagonists. This distinct pharmacological profile suggests a viable strategy for identifying 5-HT₇ receptor-mediated responses in the central nervous system. Additionally, the recently available antagonist SB-269770 can be used to further characterize a 5-HT₇-mediated response (Lovell et al., 1999; Bacon and Beck, 2000).

The distribution of 5-HT₇ receptors in the central nervous system has been determined using in situ hybridization and receptor autoradiography using tritiated 5-CT. In both the guinea pig and the rat, there is a comparable distribution between the 5-HT₇ mRNA determined by these assays (To et al., 1995; Gustafson et al., 1996; Heidmann et al., 1998). 5-HT₇ receptors are widely expressed in the brain, with their highest expression levels being found in the anterior, paraventricular, and rhomboid nuclei of the thalamus and the CA3 region of the hippocampus (Gustafson et al., 1995; To et al., 1995).

In summary, the 5-HT₇ receptor is positively linked to adenylate cyclase, is abundantly expressed in limbic regions, and displays a distinct pharmacological profile. Yet, despite these defining characteristics, its functional role in the brain remains poorly understood. Therefore, we used whole-cell patch clamp recording to examine the effect of 5-HT in the anterior thalamus, a region where 5-HT₇ receptors are abundantly expressed. In the anterodorsal nucleus of the thalamus (ADn), we found that 5-HT induces a membrane depolarization and associated inward current and that this current is signaled by stimulation of 5-HT₇ receptors. These results identify a physiological response elicited by the activation of 5-HT₇ receptors in the thalamus.

	Materials and Methods

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Preparation of Brain Slices. Male Sprague-Dawley rats (p28-p42) were anesthetized with halothane and then decapitated. The brain was removed and placed in ice-cold Ringer's solution (composition in mM: 119 NaCl, 2.5 KCl, 1.3 MgSO₄, 2.5 CaCl₂, 1 NaH₂PO₄, 26.6 NaHCO₃, and 11 glucose) bubbled to equilibrium with 95% O₂/5% CO₂. The cerebellum and the anterior forebrain were removed while the rest of the brain was affixed to a stage with cyanoacrylate glue. A vibratome was used to cut 400 µm thick brain slices containing the anterior thalamus. These slices were placed into a submerged chamber (Sakmann and Stuart, 1995) containing constantly bubbled Ringer's solution and left to recover for at least 1 h at room temperature. When recording, the slices were placed in a recording chamber (Nicoll and Alger, 1981) where they were held submerged between two nylon nets and continuously perfused (1-2 ml/min) with normal Ringer's solution bubbled with 95% O₂/5% CO₂ at 30°C_.

Electrophysiological Recordings. Whole-cell recordings were obtained from the anterodorsal (ADn) and anteroventral thalamic nuclei using the "blind" tight-seal patch clamp recording technique (Blanton et al., 1989). The thalamic nuclei were identified using visual landmarks, specifically the stria medullaris and the 4th ventricle. A Flaming/Brown horizontal puller was used to make recording pipettes from borosilicate glass, o.d. 1.2 mm (Sutter Instrument Co., Novato, CA). The pipettes were filled with either a potassium gluconate solution (composition in mM: 115 potassium gluconate, 5 NaCl, 10 HEPES, 10 EGTA, 2 MgCl₂, 1 CaCl₂, 5 ATP, 0.5 GTP) or a cesium gluconate solution (composition in mM: 100 CsOH, 100 D-gluconic acid, 5 NaCl, 10 HEPES, 10 BAPTA, 1 MgCl₂, 4 CaCl₂, 5 ATP, and 0.5 GTP. The pH of these solutions was adjusted to 7.3 or 7.4 with KOH or CsOH, respectively.

Data Analysis. Data were analyzed using Origin (Microcal Software, Northampton, MA). The equation used to describe the dose-response relationship was y = A₁ + (A₂  A₁)/1 + e^{(x  x₀)/dx}, where dx is the Hill slope, A₁ and A₂ are the minimum and maximum inward current, respectively, and x is the effective concentration (EC₅₀). Except where indicated, data are presented as means ± S.E.M. The pA₂ value for mesulergine was estimated using the following equation.
where A and A' are equieffective agonist concentrations in the absence and presence of antagonists, respectively.

Reverse Transcriptase (RT)-Polymerase Chain Reaction (PCR). The brain was removed as described previously and then manually dissected to obtain the anterior thalamic nuclei for RNA isolation. RNASTAT 60 (Tel-Test, Friendswood, TX) was used to extract RNA from the tissue according to the manufacturer's instructions. For single-cell RT-PCR, whole-cell patch clamp was obtained by the blind tight-seal technique and the cell's contents were removed by suction. The electrode tip was then broken in a tube and the contents of the electrode expelled. The tube was placed on dry ice until the RT reaction was started. The cDNA was synthesized with 20 units of avian myeloblastosis virus reverse transcriptase, 10 mM Tris, 50 mM KCl (Roche Molecular Biochemicals, Indianapolis, IN and Promega, Madison, WI), 4 µg of random hexanucleotide primers, 1 mM dNTPs, 50 units of RNase inhibitor, and sterile water to make approximately 20 to 32 µl of reaction mix. The reaction was left at 25°C for 10 min, placed at 42°C for 60 min, and then placed at 99°C for 5 min. The cDNA from the 5-HT₇ receptor was amplified using the following primer pairs: 5'-GAATATCAACCGGAAGCTCTC (sense primer A); 5'-CTGGATCATGTATCATGACCC (anti-sense primer A') (Heidmann et al., 1997); and 5'-GTGGACTTGTGGAAAGCACC (anti-sense primer C'). These two primer pairs should yield products of 132 bp for A to A' and 451 bp for A to C'. The PCR reaction was performed in a Perkin-Elmer GeneAmp PCR System 2400 (30-40 cycles of 95°C for 1 min, 60°C for 40 s, and 72°C for 1 min). The amplification of both primer pairs was done in 10 mM Tris/HCl, 50 mM KCl, 1.5 to 2.5 mM MgCl₂, 200 µM dNTP mix, 40 nmol of each primer at pH 8.3 with 5 units of Taq DNA polymerase (Roche Molecular Biochemicals/Fisher Biotech, Pittsburgh. PA). Restriction analysis was done using AvaI and SphI (New England BioLabs, Beverly, MA) according to the manufacturer's instructions.

	Results

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Effect of Serotonin in the Anterior Thalamus. To locate a serotonin response mediated by 5-HT₇ receptors, we focused on the anterior thalamus, a region where these receptors are abundantly expressed (Gustafson et al., 1995; To et al., 1995). Bath application of 5-HT (1-100 µM) elicited a membrane depolarization (Fig. 1) or inward current (Fig. 2) in the vast majority of neurons of the ADn (121 of 123 cells tested). In contrast, comparable administration of serotonin to cells of the anterior ventral thalamic nucleus resulted in less consistent results; cells exhibited either no effect, a small hyperpolarization, or a small depolarization (n = 10 cells tested). Accordingly, all experiments outlined in this paper were conducted on cells of the ADn.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Effect of serotonin in the ADn. Bath application of serotonin elicits a concentration-dependent slow membrane depolarization in a cell of the ADn. The cell resting membrane potential was 61 mV.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Serotonin induces a concentration-dependent inward current in the ADn. A, current traces illustrating the inward currents elicited by increasing concentrations of serotonin in a representative cell of the ADn. B, plot of the peak 5-HT-induced inward current for the cell illustrated in A. Vh = 60 mV, Iholding = 10 pA.

Single-Cell RT-PCR Analysis. Previous in situ studies have located 5-HT₇ receptor mRNA in the ADn, but they have not identified the cell types that expressed it (Lovenberg et al., 1993; Gustafson et al., 1995; To et al., 1995). These studies have generally assumed that neurons are the source of the 5-HT₇ message, but this has not been directly investigated. Furthermore, there is evidence that thalamic glia express 5-HT₇ receptor mRNA (Hirst et al., 1997). If 5-HT₇ receptors play a role in mediating the depolarization we observed in the ADn, then 5-HT₇ receptor mRNA must be expressed in the neurons of this nucleus. Therefore, we first tested this possibility by using single-cell RT-PCR techniques. In four separate trials, we recorded from single cells in the ADn and extracted the contents of these cells by suction, obtaining the mRNA. We next performed RT-PCR to amplify 5-HT₇ mRNA using primers chosen to amplify across an intron to eliminate contamination by genomic DNA (Fig. 3A). Figure 3, B and C, depicts results from one of nine cells in which this experiment was successful. Administration of serotonin to this cell resulted in the characteristic inward current described previously (Fig. 3B). Subsequent amplification of this cell's contents using primers A and A' resulted in a band of approximately 135-bp long, consistent with the prediction for amplification of 5-HT₇ receptor mRNA. In two additional cells, we obtained similar results when using primers A + C and A + A' in nested PCR reactions.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Single-cell RT-PCR analysis of 5-HT7 mRNA in the ADn. A, diagram of the genomic organization of the 5-HT7 receptor gene and the position of the primers used in the present experiments (redrawn after Heidmann et al., 1997). Black lines indicate exons and gray lines indicate introns. Arrows denote the approximate positions for the primers. B, inward current elicited by 5-HT in a cell of the ADn. C, agarose gel electrophoresis of the PCR products amplified using selective 5-HT7 primers (A and A') from a 5-HT7 receptor plasmid (plasmid lane), the product of an RT reaction for material "harvested" from the extracellular space in the slice (slice lane), and cDNA from the cell in B (cell lane). The right lane depicts 100-bp ladder.

Identification of the 5-HT Receptor That Signals the Membrane Depolarization in the ADn. Although our single-cell RT-PCR experiments indicated that the 5-HT₇ mRNA is produced in the cells from which we record, this did not prove the receptor mediating the membrane depolarization was of the 5-HT₇ subtype. In fact, previous in situ hybridization studies have detected expression of mRNA for several distinct serotonin receptor subtypes, including the 5-HT_1A, 5-HT_2A, and 5-HT_2C receptors, over the ADn (Gustafson et al., 1995; To et al., 1995; Wright et al., 1995). Therefore, to identify whether the 5-HT₇ receptor subtype was indeed the receptor responsible for the depolarization, we took advantage of the distinct pharmacological profile of 5-HT₇ receptors. This analysis was possible because the 5-HT-induced inward current was reproducible, concentration-dependent, and non-desensitizing.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effects of serotonergic agonists in the ADn. A, in three cells of the ADn, bath administration of 5-HT, 5-CT, and 5-MeOT induce similar inward currents. B, in contrast, bath administration of DOI was without effect, although the same cell responded to serotonin. C, administration of 8-OHDPAT elicits a small inward current and inhibits the effect of serotonin. The reduction in the response to serotonin was surmountable by the administration of a 10-fold higher concentration of 5-HT. Vh was 60 mV for all cells, Iholding ranged from 40 pA to 20 pA. All calibration bars depict 25 pA and 100 s.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Concentration-response curve for serotonin and 5-CT. In voltage clamp mode (Vh  60 mV), bath application of increasing concentrations of 5-HT induced progressively larger inward currents (n = 5 cells). The maximum current observed for serotonin at each concentration was used to fit the data to an occupational model for each cell (see Methods). This model estimated the maximal effect of serotonin at that cell, and this value was used to normalize the data. Because of the slow recovery from 5-CT, a single concentration of 5-CT was tested per cell, and the resulting values were normalized with respect to the mean response observed with a maximal concentration of 5-CT (3 µM). 5-CT was approximately 18 times more potent than 5-HT at eliciting an inward current in the ADn. The values next to each point in the curve for 5-CT indicate the number of cells tested at that concentration.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Antagonism of the serotonin-induced inward current in the ADn. A, bath administration of cyanopindolol (Cy) had no detectable effect on the ability of serotonin to induce an inward current. B through D, in contrast, administration of mesulergine, methysergide, or LSD antagonized this effect of serotonin. Vh was 60 mV for all cells; Iholding ranged from 30 pA to no holding current. All calibration bars depict 25 pA and 100 s.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Concentration-response curve for serotonin in the presence of mesulergine or cyanopindolol. Mesulergine, but not cyanopindolol, elicited a shift in the concentration-response curve for serotonin. The EC50 for serotonin was 1.7 µM (, n = 5 cells, data redrawn from Fig. 5 for illustration purposes). The EC50 for serotonin in the presence of mesulergine was 9.6 µM (, 100 nM, n = 3 cells), indicating a pA2 value of 31 nM. The EC50 for serotonin in the presence of cyanopindolol (, 10 µM, n = 3 cells) was 2.0 µM and was not different from that seen under control conditions.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   SB-269770 antagonizes the 5-HT-induced inward current. A, bath administration of the selective 5-HT7 antagonist SB-269770 (1-3 µM) antagonized the 5-HT-induced inward current. B, summary of four cells tested, indicating the inhibition of 5-HT-induced inward current in the presence of SB-260770.

	Discussion

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In this study, we have examined the possible involvement of 5-HT₇ receptors in mediating the electrophysiological effects of serotonin in the anterior thalamus. We report that neurons of the ADn express 5-HT₇ receptor mRNA as determined by single-cell RT-PCR and respond to serotonin administration with a depolarization or inward current. Based on a pharmacological analysis using selective agonists and antagonists, we conclude that this depolarization/inward current is signaled by activation of receptors of the 5-HT₇ subtype.

Single-Cell RT-PCR Analysis. Administration of serotonin elicited a membrane depolarization or inward current in the vast majority of ADn neurons examined in this study. As a first step to examine the involvement of 5-HT₇ receptors in this response, we tested for the expression of 5-HT₇ mRNA in these neurons. This was an important first step because previous studies have reported the expression of 5-HT₇ receptor-mediated responses in glial cells of the thalamus (Hirst et al., 1997). This raised the possibility that the 5-HT₇ receptor expression detected by in situ hybridization and receptor autoradiography (Gustafson et al., 1995; To et al., 1995) in this brain region may be non-neuronal. We used primers selective for the 5-HT₇ receptor to amplify cDNA obtained from RT reactions of mRNA from single cells of the ADn from which we recorded. We found that neurons that respond to bath application of 5-HT also express mRNA for the 5-HT₇ receptor. This observation was consistent with the possibility that this receptor may mediate the 5-HT-induced response. However, by in situ hybridization, several other serotonergic receptors appear to be expressed in the ADn as well, including those of the 5-HT_1A, the 5-HT_2A, and 5-HT_2C subtypes (Wright et al., 1995). This heterogeneity in serotonin receptor subtype expression led us to conduct a pharmacological analysis of the receptor mediating the 5-HT-induced depolarization and inward current in the ADn.

Pharmacological Analysis. To identify the receptor subtype involved in signaling the serotonin inward current in this area, we first tested a variety of agonists. The 5-HT-induced depolarization/inward current was concentration-dependent and mimicked by 5-CT, which was approximately 10 to 20 times more potent than 5-HT. 8-OHDPAT functioned as a low intrinsic activity partial agonist, whereas DOI was without a detectable effect on these cells. We complemented these results by examining receptor subtype-selective antagonists. We found that mesulergine, methysergide, LSD, and SB-269770 blocked the 5-HT-induced inward current whereas cyanopindolol did not. Additionally, low micromolar concentrations of ritanserin, but not ketanserin, inhibited the response. Finally, we compared the affinities of mesulergine and cyanopindolol at the receptor mediating the inward current. Mesulergine competitively antagonized the response of serotonin with an apparent affinity (pA₂) of 31 nM, whereas cyanopindolol was found to have no detectable affinity for the receptor involved.

5-HT-Induced Depolarizations. Previous studies have shown that among G-protein coupled 5-HT receptors, both 5-HT₂ and 5-HT₄ receptors can mediate slow membrane depolarizations in central neurons (North and Uchimura, 1989; Andrade and Chaput, 1991). The present results indicate that 5-HT₇ receptors now join these receptors in being capable of mediating membrane depolarizations in the central nervous system. Interestingly, it has been known for many years that an additional "orphan" serotonin receptor also mediates membrane depolarizations through a mechanism involving cAMP and up-regulation of the cation-nonselective current I_h. In a previous study in dorsal root ganglion neurons, it was suggested that this "orphan" receptor may belong 5-HT₇ subtype, but other receptor subtypes could not be excluded (Cardenas et al., 1999). In the accompanying paper, we rigorously test this hypothesis. We find that the 5-HT₇ receptor-mediated depolarization we have identified in the ADn is indeed mediated by I_h. These results identify a physiological function for 5-HT₇ receptors in the brain.