5-HT7 receptors activate the mitogen activated protein kinase extracellular signal related kinase in cultured rat hippocampal neurons

Activation of ERK in hippocampal neurons

Pergamon

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Neuroscience Vol. 102, No. 2, pp. 361±367, 2001 361 q 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/01 $20.00+0.00

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5-HT7 RECEPTORS ACTIVATE THE MITOGEN ACTIVATED PROTEIN KINASE EXTRACELLULAR SIGNAL RELATED KINASE IN CULTURED RAT HIPPOCAMPAL NEURONS M. ERRICO, a R. A. CROZIER, b M. R. PLUMMER b and D. S. COWEN a* a

Department of Psychiatry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ, USA b

Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA

AbstractÐMedications that selectively increase 5-hydroxytryptamine are currently the most commonly prescribed antidepressants. However, it is not known which receptors for 5-hydroxytryptamine, nor which post-receptor cellular signals, mediate the antidepressant actions of 5-hydroxytryptamine. The hippocampus is highly innervated by serotonergic neurons and appears to be an ideal region of the brain for studying the antidepressant role of 5-hydroxytryptamine. Treatment with antidepressants has been shown to cause increased expression of proteins in the hippocampus that appear to be protective against stress-induced atrophy. This suggests a role for pathways, such as mitogen-activated protein kinase, that regulate protein synthesis. In the present study we found that 5-HT7 receptors, expressed by cultured rat hippocampal neurons, couple to stimulation of the mitogen-activated protein kinase extracellular signal-regulated kinases ERK1 and ERK2. The 5-HT1/7 receptor-selective agonist 5-carboxamidotryptamine maleate (5-CT) as well as the 5-HT1A/7 receptor-selective agonists 8hydroxy-N,N-dipropyl-aminotetralin (8-OH-DPAT) and N,N-dipropyl-5-carboxamidotryptamine maleate (dipropyl-5-CT) were found to activate extracellular signal-regulated kinase with equal ef®cacy to 5-HT. However, the EC50 for 8-OH-DPAT was approximately 200-fold greater than that of 5-HT, a difference in potency consistent with the pharmacology of 5-HT7, but not 5-HT1A, receptors. Additionally, pretreatment with pertussis toxin, which would be expected to block the actions of 5-HT1, but not 5-HT7, receptors caused no inhibition. 4-Iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]N-2-pyridinylbenzamide hydrochloride (p-MPPI) and N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cyclohexanecarb oxamide maleate (WAY-100635), antagonists selective for 5-HT1A receptors, similarly caused no inhibition of the activity of 5-HT. In summary, these studies are the ®rst to demonstrate that 5-hydroxytryptamine activates the mitogen-activated protein kinase ERK in primary neuronal cultures. That 5-HT7 receptors couple to activation of extracellular signal-regulated kinase in hippocampal neurons suggests a possible role for 5-HT7 receptors in mediating some of the actions of antidepressants that increase 5-hydroxytryptamine. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: serotonin, 5-HT, 5-HT7 receptors, neurons.

mediate these actions. At least 16 types of mammalian receptors for serotonin have been reported. 14,24 They have been classi®ed into seven families. All are G protein-coupled receptors with the exception of 5-HT3 receptors, which are ligand-gated ion channels. For example, 5-HT1 receptors couple to G proteins of the Gi class, and couple negatively to adenylyl cyclase. 14,24 In contrast, 5-HT4, 5-HT6, and 5-HT7 receptors couple to Gs and increase levels of cAMP. 14,24 The hippocampus appears to be an ideal brain-region for studying potential mechanisms for 5-HT-mediated antidepressant treatment. Hippocampal neurons have been found to express multiple types of receptors for 5HT, including 5-HT1A, 17,29,31 5-HT2A/C, 29 and 5-HT7 23,29 receptors. Additionally, these neurons have been found to be highly sensitive to the elevated levels of glucocorticoids associated with stress and depression. In fact, major depression 2 and Cushing's syndrome (a disorder in which cortisol is elevated) 25 have been associated with a reduction in hippocampal volume. It has been proposed that decreased expression of brain-derived neurotrophic

Determining the mechanism of action of current antidepressants would greatly facilitate the synthesis of newer, more effective medications. Since many of the most commonly prescribed antidepressants selectively increase levels of serotonin (5-hydroxytryptamine or 5HT), it appears that increases in 5-HT are suf®cient to treat depression. However, it is not clear which receptors for 5-HT are required, and which cellular pathways *Corresponding author. Tel: 11-732-235-8576; fax: 11-732-357677. E-mail address: [email protected] (D. S. Cowen). Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; 5-CT, 5-carbamidotryptamine meleate; EDTA, ethylediaminetetra-acetate; ERK, extracellular signal-regulated kinase; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; 5-HT, 5-hydroxytryptamine; MAP kinase, mitogenactivated protein kinase; NGF, nerve growth factor; 8-OH-DPAT, 8-hydroxy-N,N-dipropyl-5-carboxamidotryptamine maleate; pMPPI, 4-Ido-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]N-2pyridinyl-benzamide hydrochloride; WAY-100635, N-[2-[4-(2Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cyclohexanecarb oxamide maleate. 361

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factor (BDNF) may be at least partially responsible for this atrophy. 7 Interestingly, treatment with antidepressants has been found to cause an increase in mRNA levels of BDNF and its tyrosine kinase receptor, TrkB. 19 The expression of additional proteins, including cAMP response element binding protein (CREB), are also increased by antidepressant treatment. 19 These ®ndings suggest that synthesis of select proteins may mediate the actions of antidepressants. In the present study we examined whether 5-HT receptors, expressed by hippocampal neurons, couple to activation of the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinases ERK1 and ERK2. These kinases have been shown to phosphorylate a number of transcription factors including c-Jun, p62 TCF/ Elk-1, c-Fos and c-Myc, and also appear to regulate translation of mRNA. 6 The neurotrophins nerve growth factor (NGF) and BDNF have both been shown to activate ERK. 12,20,22 Signi®cantly, ERK appears to be required for normal neuronal function. In fact, it appears to have a role in neurotrophin-stimulated neuronal differentiation and neuroprotection. 12,20 EXPERIMENTAL PROCEDURES

Materials 5-HT, 5-carboxamidotryptamine maleate (5-CT), N,N-dipropyl5-carboxamidotryptamine maleate (dipropyl-5-CT), (^)8hydroxy-N,N-dipropyl-2-aminotetralin hydrobromide (8-OHDPAT), 4-Iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]N-2-pyridinyl-benzamide hydrochloride (p-MPPI), N-[2-[4-(2Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY-100635), and Protein A:sepharose were purchased from Sigma (St Louis, MO). Pertussis toxin was purchased from Calbiochem (San Diego, CA). Cell culture Hippocampal neuronal cultures were obtained as previously described. 16 Hippocampi were isolated from embryonic day 18 Sprague±Dawley rats obtained from Taconic (Germantown, NY), and 10 6 cells were plated per poly(d-lysine) coated 35mm Petri dish. Cells were maintained in serum-free medium consisting of a 1:1 (vol/vol) mixture of Ham's F-12 and Eagle's minimal essential medium, supplemented with 25 mg/ml insulin, 100 mg/ml transferrin, 60 mM putrescine, 20 nM progesterone, 30 nM selenium, 6 mg/ml glucose, 0.5 units/ml penicillin, and 0.5 mg/ml streptomycin at 378C (95% air, 5% CO2). Cells remained in culture for 12±16 days before use. A previously described stable transfected CHO cell line expressing human 5-HT1A receptors at a density of 3000 fmol/mg membrane protein, 5,18 was maintained in medium containing Ham's F-12 Nutrient Mixture with l-glutamine, 10% dialyzed fetal bovine serum (dialyzed in membranes with 1000 Dalton molecular weight cutoffs against a 100-fold greater volume of 150 mM NaCl to remove endogenous 5-HT), 1% penicillin-streptomycin, and 400 mg/ml geneticin at 378C (95% air, 5% CO2). Cells were grown overnight under serum-free conditions before use. Assay of mitogen-activated protein kinase activity MAP kinase activity was measured following immunoprecipitation, essentially as previously described. 5,8 Some modi®cations were made in order to compensate for the small number of hippocampal cells available for immunoprecipitation. Cells were stimulated with the speci®ed concentrations of agonists for 5 min, and lysed. Total ERK1 and ERK2 were immunoprecipitated overnight from 20±60 mg cytosolic protein with 2 mg rabbit polyclonal IgG recognizing ERK1/ERK2 (C-14; Santa Cruz

Biotechnology; Santa Cruz, CA) coupled to Protein A:sepharose. Activity was measured by incorporation of 32P into 80 mg of a nine amino acid MAP kinase substrate peptide containing amino acids 95±98 of myelin basic protein (Upstate Biotechnology, Lake Placid, NY). The kinase reactions were performed at room temperature for 60 min. After centrifugation at 13 000 g for 5 min, supernatant was spotted on P81 paper and the paper washed extensively with 0.75% phosphoric acid and acetone. Incorporation of 32P into substrate peptide was measured in dried papers by scintillation spectrometry. Immunoblots Monoclonal anti-phospho-ERK1/ERK2 (Thr202/Tyr204-#9106) was obtained from New England Biolabs (Beverly, MA). Rabbit polyclonal total ERK1/ERK2 (#C14) and horseradish peroxidaseconjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cells were stimulated with the speci®ed concentrations of agonists, and lysed with a 26-gauge needle in 25 mM HEPES (pH 7.4), 50 mM NaF, 5 mM EDTA, 1 mM sodium orthovanadate, 100 mM phenylmethysulfonyl ¯uoride, 0.1% aprotinin, and 10 mg/ml leupeptin. Total ERK was immunoprecipitated overnight from approximately 20± 60 mg cytosolic protein with 2 mg rabbit polyclonal IgG recognizing ERK1/ERK2 (C-14). Proteins were separated on 12% resolving gels (Bio-Rad Laboratories, Hercules, CA) and transferred to 0.45 mm Immobolin-P polyvinylidene di¯uoride membranes (Millipore Corporation, Bedford, MA). Membranes were blocked overnight with 3% powdered milk before incubation with primary and secondary antibodies. Bound antibodies were visualized using Enhanced Luminol Chemiluminescence Reagent (NEN Life Sciences, Boston, MA) and exposure to a Kodak Image Station 440CF with a cooled, full-frame-capture CCD camera (Kodak). Net intensity of bands was calculated using Kodak Digital Science 1D Image Analysis Software (version 3.0.2) on de®ned regions of interest. Calculations were made from images captured directly by the CCD camera, avoiding the problem of saturation of signal intensity inherent in ®lm. Statistical analyses were performed using GraphPad InStat (version 2.04) with statistical signi®cance de®ned as P , 0.05. All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scienti®c data. Experiments conformed to NIH guidelines on the ethical use of animals. RESULTS

Activation of mitogen activated protein kinase by 5hydroxytryptamine and selective agonists for 5-HT1 and 5-HT7 receptors Studies of the coupling of 5-HT receptors to the MAP kinases ERK1 and ERK2 were performed in cultures of fetal (E18) hippocampal neurons. Activation of ERK was measured by two methods: (i) directly in kinase assays; and (ii) indirectly by detection of double phosphorylation of the kinase at threonine 202 and tyrosine 204Ða requirement for activation. 4 The indirect method of assaying MAP kinase activity was found to be more sensitive. This was an advantage in our studies of hippocampal neurons, in which the number of available cells was necessarily limited. Treatment of cultured hippocampal neurons with 5-HT was found to stimulate phosphorylation of ERK (Fig. 1A). Maximal stimulation varied between individual hippocampal cell preparations, and ranged from 2.5- to 10-fold increases in the level of phosphorylated kinase. Phosphorylation was found to be maximal within 2 min of treatment with 5-HT, and to remain increased for over

Activation of ERK in hippocampal neurons

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Fig. 2. 5-HT- and 5-CT-stimulate activation of ERK with similar potencies. Hippocampal neurons were incubated for 5 min with the indicated concentrations of (A) 5-HT or (B) 5-CT and lysed. Immunoprecipitated ERK1 and ERK2 were analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total ERK). Net intensities of p-ERK and Total ERK bands (intensity) were calculated as described in the Experimental Procedures, and are expressed £ 10 3. Immunoblots are representative of three separate experiments performed in duplicate. Fig. 1. 5-HT and agonists for 5-HT1/7 receptors stimulate phosphorylation of the MAP kinases ERK1 and ERK2. Hippocampal neurons were incubated for 5 min with (A) 10 mM 5-HT, 10 mM 5-CT, 10 mM dipropyl-5-CT, or 10 mM 8-OH-DPAT (DPAT) or (B) 1 mM 5-HT for the indicated times and lysed. Immunoprecipitated ERK1 and ERK2 were analysed by immunoblotting with antibody to phospho-ERK1/ ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total ERK). Net intensities of p-ERK and Total ERK bands (intensity) were calculated as described in the Experimental Procedures, and are expressed £ 10 3. Immunoblots are representative of three separate experiments performed in duplicate.

30 min (Fig. 1B). The EC50 (Fig. 2A) was consistent between preparations, and was approximately 10 nM (calculated by non-linear regression analysis of the net intensities of bands from multiple experiments). 5-CT, an agonist selective for 5-HT1 and 5-HT7 receptors, was found to stimulate activation of ERK with ef®cacy equal to 5-HT. Additionally, 8-OH-DPAT and dipropyl-5-CT, two agonists selective for 5-HT1A and 5-HT7 receptors, were found to stimulate similar levels of maximal activation. This was best seen in studies where the ef®cacies of agonists were directly compared in the same preparation of neurons (Fig. 1A). As mentioned above, multiple types of 5-HT receptors are expressed in the hippocampus. We chose not to examine

the potential coupling of each of these receptors to the activation of MAP kinase. Instead, because 5-CT, 8-OHDPAT, and dipropyl-5-CT were found to stimulate large increases in activated ERK, we focused on determining whether the actions of these agonists were mediated by 5HT1A or 5-HT7 receptors. Although the pharmacology of 5-HT1A and 5-HT7 receptors is similar, the relative potencies of agonists can be used to differentiate receptor subtypes. In our studies the EC50 for 5-CT was found to be similar (10 nM) to that of 5-HT (Fig. 2B). However, the estimated EC50 for 8-OH-DPAT (Fig. 3A) was found to be 200-fold greater than that for 5-HT (2 mm vs 10 nM). Similar differences in potency for 5HT relative to 8-OH-DPAT were observed when MAP kinase activity was directly measured (Fig. 3B). This pharmacology is consistent with 5-HT7, but not 5HT1A, receptors. 1,10,13,14,21,23,31 Lack of inhibition by antagonists selective for 5-HT1A receptors The effect of p-MPPI and WAY-100635, antagonists selective for 5-HT1A receptors, relative to 5-HT7 receptors 13,30 were tested. Neither antagonist was found to

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M. Errico et al. Table 1. Antagonists For 5-HT1A receptors inhibit the actions of 5-HT in a cell line expressing 5-HT1A receptors, but not in hippocampal neurons Cell type

Treatment

Net intensity (% basal) Mean ^ S.E.M. (P-value)

Hippocampal neurons (n ˆ 9)

100 nM 5-HT 100 nM 5-HT 1 10 mM p-MPPI 100 nM 5-HT 1 10 mM WAY-100635

382 ^ 60 (2) 505 ^ 127 (n.s.) 539 ^ 96 (n.s.)

CHO cell line (n ˆ 6)

100 nM 5-HT 100 nM 5-HT 1 1 mM p-MPPI 100 nM 5-HT 1 1 mM WAY-100635

17 643 ^ 1833 (2) 250 ^ 96 (P , 0.001) 258 ^ 101 (P , 0.001)

Hippocampal neurons or transfected CHO cells expressing 5-HT1A receptors were incubated for 5 min with 100 nM 5-HT in the presence or absence of the speci®ed concentrations of p-MPPI or WAY-100635 and lysed. Antagonists were added immediately before 5-HT. Immunoprecipitated ERK1 and ERK2 were analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Net intensities of p-ERK bands were calculated, as described in the Experimental Procedures, from three or more separate experiments and the means ^ S.E.M. expressed as % basal net intensity of p-ERK. P-value vs 5-HT in the absence of antagonists, repeated measures ANOVA. n.s., not statistically signi®cant (P . 0.05). Table 2. Pertussis toxin inhibits the actions of 5-HT in a transfected cell line expressing 5-HT1A receptors, but not in hippocampal neurons Cell type

Treatment

Net intensity (% basal) Mean ^ S.E.M. (P-value)

Hippocampal neurons (n ˆ 4)

1 mM 5-HT 600 ng/ml pertussis toxin 1 mM 5-HT 1 600 ng/ml pertussis toxin

342 ^ 71 (2) 86 ^ 27 (P , 0.05) 296 ^ 73 (n.s.)

CHO cell line (n ˆ 6)

1 mM 5-HT 150 ng/ml pertussis toxin 1 mM 5-HT 1 150 ng/ml pertussis toxin

15 683 ^ 1086 (2) 100 ^ 1 (P , 0.001) 100 ^ 1 (P , 0.001)

Hippocampal neurons or transfected CHO cells expressing 5-HT1A receptors were incubated for 5 min with 1 mM 5-HT after pre-incubation for 6 h with the indicated concentrations of pertussis toxin and then lysed. Immunoprecipitated ERK1 and ERK2 were analysed by immunoblotting with antibody to phospho-ERK1/ ERK2 (p-ERK). Net intensities of p-ERK bands were calculated, as described in the Experimental Procedures, from three or more separate experiments and the means ^ S.E.M. expressed as % basal net intensity of p-ERK. P-value vs 5-HT in the absence of toxin, repeated measures ANOVA. n.s., not statistically signi®cant (P . 0.05).

inhibit the phosphorylation of ERK stimulated by 5-HT in hippocampal neurons (Table 1). This was despite 100-fold greater concentrations of antagonists than 5HT. In fact, both antagonists appeared to cause a small (although not statistically signi®cant) increase in activation of ERK above that seen with 5-HT, in the absence of antagonists. The lack of inhibition was in contrast to our ®ndings in studies of cell lines expressing 5-HT1A receptors. We have previously reported that 5-HT stimulates activation of ERK in a stable transfected CHO cell line, expressing 5-HT1A receptors at a density of 3000 fmol/mg membrane protein. 5,18 In those cells, 5-HT caused a very large (176-fold) increase in the level of phosphorylated ERK (Table 1). This increase was completely inhibited by both p-MPPI and WAY-100635. Signi®cantly, inhibition was complete when antagonists were included with 5-HT at only 10-fold greater concentrations. Lack of inhibition by pre-treatment with pertussis toxin Neurons were pre-treated with pertussis toxin in order to further demonstrate that the activation of ERK stimulated by 5-HT was mediated by 5-HT7, and not 5-HT1, receptors. Pertussis toxin would be expected to uncouple all subtypes of 5-HT1 receptors from required Gi-type G

proteins, but not 5-HT7 receptors from Gs. Pre-treatment of hippocampal neurons with high concentrations of toxin (600 ng/ml for 6 h) caused no inhibition of the activation stimulated by 5-HT (Table 2). In contrast, pre-treatment of the CHO cell line with only 150 ng/ml pertussis toxin for 6 h caused complete inhibition of the activation of ERK stimulated by 5-HT1A receptors. DISCUSSION

We found that 5-HT activates MAP kinase in hippocampal neurons. The magnitude of maximal activation varied somewhat between hippocampal preparations but averaged approximately four-fold. This experimental variability probably re¯ected minor differences between preparations, as well as heterogeneity in the percentage of cells expressing 5-HT7 receptors. Signi®cantly, in studies directly comparing the ef®cacies of the 5-HT1/7 selective agonist 5-CT and the 5-HT1A/7 selective agonists 8-OH-DPAT and dipropyl-5-CT maleate, in the same neuronal preparations, all were found to activate ERK with ef®cacies equal to 5-HT. However, 5-CT and 5-HT were found to be much more potent agonists than 8-OHDPAT. This pharmacology for activation of MAP kinase in

Activation of ERK in hippocampal neurons

Fig. 3. 8-OH-DPAT activates ERK with low potency relative to 5-HT. (A) Hippocampal neurons were incubated with the indicated concentrations of 8-OH-DPAT (DPAT) for 5 min and lysed. Immunoprecipitated ERK1 and ERK2 were analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total ERK). Net intensities of p-ERK and Total ERK bands (intensity) were calculated as described in the Experimental Procedures, and are expressed £ 10 3. Immunoblots are representative of ®ve separate experiments performed in duplicate. (B) Hippocampal neurons were incubated with the indicated concentrations of 5-HT or 8-OH-DPAT (DPAT) for 5 min and lysed. The activity of immunoprecipitated ERK1 and ERK2 was measured as described in the Experimental Procedures. The data represent the means ^S.E.M. of ®ve separate experiments (for each agonist), each performed in triplicate, with activity expressed as a percentage of basal activity.

hippocampal neurons is consistent with mediation by 5HT7 receptors, but not 5-HT1A receptors. 1,10,13,14,21,23,31 Also consistent with activation of ERK by 5-HT7 receptors was a lack of inhibition by p-MPPI and WAY-100635, two selective antagonists for 5-HT1A receptors. Inclusion of either antagonist with 5-HT, at 100-fold greater concentrations, caused no inhibition of the activation of MAP kinase stimulated by 5-HT. This was in contrast to our ®ndings in studies of transfected CHO cells expressing 5-HT1A receptors. It should be noted that the activation of MAP kinase stimulated in the CHO cells (176-fold) was much greater in magnitude than that seen in hippocampal cells (average of fourfold). Additionally, the density of 5-HT1A receptors expressed by the transfected cells was much greater than the density of 5-HT1A receptors that would be expected to be expressed by hippocampal cells. The CHO cells express receptors at a density of 3000 fmol/ mg membrane protein 5,18 in contrast to the 200 fmol/mg

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membrane protein expressed in the hippocampus of adult rats. 3 Therefore, if the activation of ERK in hippocampal neurons had been mediated by 5-HT1A receptors, one would have expected that lower concentrations of antagonists would have been required in neuronal studies than in the CHO cell studies. In contrast, 100-fold greater concentrations of antagonist than 5-HT failed to cause inhibition in the hippocampal studies, yet 10-fold greater concentrations of antagonist than 5-HT caused complete inhibition in the CHO cell studies. Further evidence for mediation by 5-HT7 and not 5HT1A receptors was provided by the lack of effect of pertussis toxin in hippocampal studies. Pertussis toxin selectively ribosylates G proteins of the Gi/o class, causing uncoupling of receptor from G protein. All subtypes of 5-HT1 receptors utilize Gi/o, whereas 5-HT7 receptors couple to activation of adenylyl cyclase 1,23 and are consequently thought to utilize Gs. In our studies, high concentrations of pertussis toxin (600 ng/ml for 6 h) caused no inhibition of 5-HT-stimulated activation of ERK in hippocampal neurons. In contrast, treatment with only 150 ng/ml pertussis toxin caused complete inhibition in transfected CHO cells expressing 5-HT1A receptors. It should be noted that higher concentrations of toxin were not tested in the neuronal studies due to concern that any potential inhibition resulting from higher concentrations might be the result of non-G proteinmediated effects. In fact, in preliminary experiments with neurotrophins, we found that treatment with 600 ng/ml pertussis toxin, for 6 h, caused a 34% inhibition of the activation of ERK stimulated by BDNF. This inhibition is likely to be non-selective since BDNF acts via a receptor tyrosine kinase, and not a G-protein coupled receptor. Although our ®ndings demonstrate activation of MAP kinase by 5-HT7 receptors, they do not rule out activation of MAP kinase in hippocampal neurons by other receptors for 5-HT. Neurons in the hippocampus express multiple receptors for 5-HT, in addition to 5-HT7 receptors. These include 5-HT1A17,29,31 and 5-HT2A/C receptors. 29 We 5,18 and others, 9 have previously reported, and demonstrate again in the present studies, that transfected 5-HT1A receptors can couple to activation of MAP kinase. Additionally, 5-HT2 receptors have been reported to activate MAP kinase in smooth muscle cells and transfected cell lines. 11,15,28 We have not performed experiments (e.g. binding assays) to determine which 5-HT receptors are expressed by our cultured neurons. It is possible that the conditions in which our neurons were isolated and cultured may have inadvertently selected for neurons that express 5-HT7 receptors over neurons that express other receptors for 5-HT. While the effect of cAMP on MAP kinase activity has been shown to be cell-dependent, it is clear that compounds which increase cAMP can activate ERK in neurons. In cerebellar granule cells, for example, it has been shown that pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and forskolin stimulate ERK1 and ERK2 through a cAMP (protein kinase A)-dependent pathway. 26 Activation of MAP kinase, by cAMP, appears to require B-Raf, a Raf isoform expressed in neurons.

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B-Raf can be activated by the small molecular weight G protein Rap1, which can be activated by protein kinase A. 27 Therefore, the proposed pathway for activation of ERK by 5-HT7 receptors would be as follows: (i) agonists stimulate increases in cAMP; (ii) which cause activation of protein kinase A; (iii) which activates Rap1; (iv) which activates B-Raf; (v) which activates MAP kinase kinase (MEK); (vi) which activates ERK. That 5-HT7 receptors, endogenously expressed by hippocampal neurons, activate MAP kinase, suggests a possible,

previously unidenti®ed, role for 5-HT7 receptors in similarly mediating some of the antidepressant actions of 5-HT.

AcknowledgementsÐThese studies were supported by a NARSAD Young Investigator Award, a grant from the Foundation of the University of Medicine and Dentistry of New Jersey, and by NIH grants MH60100 (D.S.C.) and HD23315 (M.R.P.)

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