Genetic knockout and pharmacological blockade studies of the 5-HT7 receptor suggest therapeutic potential in depression

Neuropharmacology 48 (2005) 492–502 www.elsevier.com/locate/neuropharm

Genetic knockout and pharmacological blockade studies of the 5-HT7 receptor suggest therapeutic potential in depression M. Guscotta,1,), L.J. Bristowb,1, K. Hadinghama, T.W. Rosahla, M.S. Beera, J.A. Stantona, F. Bromidgea, A.P. Owensa, I. Huscrofta, J. Myersa, N.M. Rupniaka, S. Patela, P.J. Whitinga, P.H. Hutsona, K.C. Fonec, S.M. Biellod, J.J. Kulagowskia, G. McAllistera a

Neuroscience Research Centre, Merck, Sharp & Dohme Research Laboratories, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK b Merck Research Laboratories, MRLSDB1, 3535 General Atomics Court, San Diego, CA 92121, USA c School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, NG7 2UH Nottingham, UK d Biological Timing Laboratory, Department of Psychology, 58 Hillhead Street, University of Glasgow, G12 8QB Scotland, UK Received 30 June 2004; received in revised form 19 October 2004; accepted 27 November 2004

Abstract The affinity of several antidepressant and antipsychotic drugs for the 5-HT7 receptor and its CNS distribution suggest potential in the treatment of psychiatric diseases. However, there is little direct evidence of receptor function in vivo to support this. We therefore evaluated 5-HT7 receptors as a potential drug target by generating and assessing a 5-HT7 receptor knockout mouse. No difference in assays sensitive to potential psychotic or anxiety states was observed between the 5-HT7 receptor knockout mice and wild type controls. However, in the Porsolt swim test, 5-HT7 receptor knockout mice showed a significant decrease in immobility compared to controls, a phenotype similar to antidepressant treated mice. Intriguingly, treatment of wild types with SB-258719, a selective 5-HT7 receptor antagonist, did not produce a significant decrease in immobility unless animals were tested in the dark (or active) cycle, rather than the light, adding to the body of evidence suggesting a circadian influence on receptor function. Extracellular recordings from hypothalamic slices showed that circadian rhythm phase shifts to 8-OH-DPAT are attenuated in the 5-HT7 receptor KO mice also indicating a role for the receptor in the regulation of circadian rhythms. These pharmacological and genetic knockout studies provide the first direct evidence that 5-HT7 receptor antagonists should be investigated for efficacy in the treatment of depression. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: 5-HT7 receptor; 5-HT7 receptor knockout mouse; 5-HT7 receptor antagonist; Forced swim test; Depression; Circadian rhythms

1. Introduction

) Corresponding author. Tel.: C44 1279 440566; fax: C44 1279 440369. E-mail address: [email protected] (M. Guscott). 1 MG and LJB contributed equally to this work. 0028-3908/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.11.015

The 5-HT7 receptor is one of 14 distinct receptor subtypes thought to mediate the diverse range of CNS effects of the neurotransmitter, serotonin (reviewed in Barnes and Sharp, 2000). The 5-HT7 receptor is the most recent addition to the 5-HT receptor family having been cloned from a variety of species, including man, rat,

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mouse and guinea-pig (Plassat et al., 1993; Ruat et al., 1993; Shen et al., 1993; Tsou et al., 1994; Gelernter et al., 1995). The 5-HT7 receptor exhibits only low (40%) homology with other Gs coupled 5-HT receptors and is encoded by a single gene on human chromosome 10 (Krobert et al., 2001). Although it is found in four different isoforms (5-HT7a, 5-HT7b, 5-HT7c, and 5-HT7d), only two (5-HT7a and 5-HT7b) are present in both rat and human, and there appears to be no pharmacological differences between them (Gustafson et al., 1996). In situ hybridisation studies revealed CNS expression in amygdala, cortex, hippocampus, thalamus, septum, hypothalamus and suprachiasmatic nucleus (Gustafson et al., 1996). 5-HT7 receptors are defined pharmacologically by their high affinity for 5-CT, 5-HT, 5-MeOT and methiothepin, moderate affinity for 8-OH-DPAT and ritanserin and low affinity for pindolol, sumatriptan and buspirone (Sleight et al., 1995). This CNS distribution, coupled with the receptor’s relatively high affinity for several psychoactive drugs, including hallucinogenics (e.g. LSD), antipsychotics (e.g. clozapine) and antidepressants (e.g. amitriptyline), have implicated the 5-HT7 receptor as a potential therapeutic target in psychosis and depression. In support of this, chronic antidepressant treatment down regulates 5-HT7 receptor expression in rat hypothalamus (Mullins et al., 1999). Recent evidence also suggests that 5-HT7 receptors may be involved in the regulation of circadian rhythms (Lovenberg et al., 1993; Ehlen et al., 2001). Thus in rat SCN slices, 8-OH-DPAT-induced phase advances in spontaneous cell firing were attenuated by ritanserin, but not pindolol, suggestive of a 5-HT7 receptor pharmacology (Lovenberg et al., 1993; Thomas et al., 1998). However, until now little direct evidence of receptor function in vivo has been available to support any of these proposals. In this study, we have examined the in vivo role of the 5-HT7 receptor in mice in which the receptor gene has been deleted by testing the mice in a range of behavioural assays sensitive to potential psychotic states. In addition, where a behavioural phenotype was observed, we also determined the effect of treating the wild type mice with the selective 5-HT7 receptor antagonist, SB-258719 (Forbes et al., 1998; Lovell et al., 2000). The data from both approaches provide the first, direct, preclinical validation of the 5-HT7 receptor as a therapeutic target for depression and add further support to its potential role in circadian/sleep disorders.

2. Materials and methods 2.1. Compound synthesis SB-258719 was synthesised and used for behavioural studies. (R)-[35S]-1-[2-(1-benzenesulfonyl-pyrrolidin-

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2-yl)-ethyl]-4-methyl-piperidine (termed compound A) was made following a reported generic synthetic route for such compounds (Collinson et al., 2002), and confirmed to be a high affinity 5-HT7 receptor antagonist suitable for use in autoradiography experiments. Desipramine HCl (DMI; 20 mg/kg; i.p.; Sigma–Aldrich, Poole, U.K.) was dissolved in 0.9% saline and administered in a dosing volume of 10 ml/ kg, 30 min prior to testing. All doses are expressed as freebase.

2.2. Generation and characterisation of 5-HT7 receptor (Htr7ÿ/ÿ) mice A DNA probe, corresponding to DNA nucleotides 109–673 of the published sequence (Ruat et al., 1993) was generated by PCR using oligonucleotides 5#-ATGATGGACGTTAACAGCAGC-3# and 5#-GGTGATCCCAAGGTACCTGTC-3# and whole mouse brain cDNA as template. This probe was used to screen a BAC library (Research Genetics Inc.) containing mouse 129Sv strain genomic sequences, and a single positively hybridising BAC clone identified. From the insert DNA from this clone, subclones were generated containing 9 kb XbaI and 6.5 kb HindIII/BglII fragments, containing exon 1 of the mouse 5-HT7 receptor gene. As shown in Fig. 1a, a targeting vector was then constructed by insertion of the 1.3 kb short and 5.2 kb long arms into the previously described cassette vector pBS246-neo-tk-1 (McKernan et al., 2000) and transfected into AB2.2 (Lexicon Genetics, Woodlands, Texas) embryonic stem cells as described previously (Crawley and Paylor, 1997). Homologous recombinants were identified by Southern blotting using a probe external to the construct, these being identified by the presence of a 3-kb hybridising band in addition to the WT band of 9.1 kb after digestion with BamHI (Fig. 1b). Correctly targeted ES cell clones were microinjected into C57Bl/6 blastocysts, and three of the six clones injected gave rise to highly chimeric males, which transmitted the targeted mutation in their germline, as determined by PCR using primers flanking the short arm on tail DNA (P1 and P2, Fig. 1). Heterozygous mice (F1 generation) were crossed and wild type (WT) with WT and homozygous (HO) with HO offspring (F2 generation) were mated using a randomized breeding strategy to generate WT and HO (F3 generation) mice for experimental use. Mice were genotyped using oligonucleotides P1 5#-CTTCAAGTTGTCATTTGCATGTGT-3# and P2 5#-GGATGCGGTGGGCTCTATGGCTTCTGA-3# to detect the 1.3-kb long targeted PCR product and P1 and P4 5#-GCAACTGCTTGGTGGTGATCTCCG-3# to detect the 1.1-kb long wild type PACER product (Fig. 1c).

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Fig. 1. Gene targeting and validation of the 5-HT7 receptor (Htr7ÿ/ÿ) knockout mice; (a) schematic diagram of targeting construct; red: probe used for genomic Southern blot; blue: short and long arm of targeting vector; black: exon 1 of 5-HT7 receptor gene; P1–P3: PCR primers used for genotyping. (b) Southern blotting result showing five wild type (WT) and one correctly targeted (HE) ES cell clone, which was used to establish knockout mouse line. (c) Genotyping examples using the PCR primers described in Section 2 and (a). Samples of WT mice gave a 1.1-kb only (bottom panel) and of homozygous (HO) mice a 1.3-kb long PCR product only (top panel) and (d) autoradiography of [35S]compound A, a high affinity 5-HT7 receptor antagonist demonstrating binding to the thalamus (T) of wild type but not knockout mouse brain sections.

2.3. Mouse brain autoradiography Mice were euthanized, the brains removed and snap frozen in isopentane at ÿ35  C. Coronal sections (10 mm) were cut on a microtome-cryostat at ÿ17  C, thaw mounted onto ‘‘superfrost plus’’ microscope slides (BDH), dried and stored at ÿ70  C. Thawed sections were pre-incubated at RT for 30 min in 170 mM TRIS buffer, pH 7.6 (plus 4 mM CaCl2, 0.01% ascorbate, 10 mM pargyline), followed by a 60-min incubation, at RT, in the same buffer containing 3 nM [35S]compound A G 10 mM SB-258719. Finally, slides were washed for 5 min in cold TRIS buffer, briefly dipped in deionized water and dried under a stream of cold air before storage overnight at 4  C. Slides were then exposed to Hyperfilm Betamax (Amersham) for 48 h. Autoradiograms were quantified using an MCID image analyser

(Imaging Research, Canada). Relative optical densities (ROD) were determined (minus background ROD levels) in the absence (total) and presence (non-specific) of SB-258719.

2.4. Behavioural studies Behavioural assays were performed on male wild type and knockout mice (background Z 50% 129SvEv/50% C57Bl/6J) aged approximately 10 weeks at the start of the study. They were maintained on a 12-h light:dark cycle (lights on at 07:00 hours) with food and water freely available. Dark cycle experiments were carried out 1–6 h after lights out. All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act (1986).

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2.5. Neurological screen This set of tests was performed to give a rapid assessment of gross motor and body temperature phenotype changes (Crawley and Paylor, 1997). Rectal temperature: core body temperature was electronically measured (‘Physitemp’ thermometer model BAT-12) in mice using a rectal probe (‘Physitemp’, model 0024). Rotarod: the mice were placed on a murine rotarod revolving at a constant 16 rpm (Acceler rotarod for mice; Jones and Roberts, model 7650). The time that they were able to stay on the rotarod was measured up to a maximum of 120 s. Beam balance: mice were placed individually in the centre of one of four beams (length Z 60 cm) of different cross-sectional dimensions and made of different materials: (a) square metal beam (0.9 cm diameter), (b) round wooden beam (0.8 cm diameter), (c) round metal beam (0.8 cm diameter), and (d) round metal beam (0.4 cm diameter). The time that the mice were able to remain on the beams was measured up to a maximum of 120 s. Swim test: mice were placed at one end of a 60-cm long swim tank containing 8–10 cm water (24–25  C). A visible platform (12 cm ! 8 cm) was placed at one end of the tank. The latency to reach the visible platform at the other end was measured. 2.6. Prepulse inhibition Startle responses were recorded using eight SR-LAB stabilimeter chambers (San Diego Instruments, San Diego, CA). The mice were exposed to white noise (65 dB) for at least an hour before use. The mice then entered the stabilimeter chambers and after a 5-min acclimation period (background noise Z 65 dB) they were exposed to 5 ! 120 dB tones (40 ms duration, 30 s intertrial interval (ITI)). These were habituation tones to partially habituate the mice to receiving tones and were not used in the statistical analysis. This was followed by random presentations of 10 each of seven different stimuli: ‘pulse’ – 120 dB, 40 ms; ‘no stim’ – a 60-ms recording only; ‘pp75’ – 75 dB, 40 ms; ‘pp80’ – 80 dB, 40 ms; ‘pp70p’ – 70 dB prepulse followed by a 120-dB pulse, 100 ms after the start of the prepulse; ‘pp80p’ – 80 dB prepulse followed by a 120-dB pulse, 100 ms after the start of the prepulse; ‘pp90p’ – 90 dB prepulse followed by a 120-dB pulse, 100 ms after the start of the prepulse. The mean ITI was 15 s (range 11–20 s). The mean startle amplitude from a 60-ms recording window initiated at the start of the pulse (or prepulse in prepulse alone trials) was calculated for each group. 2.7. Porsolt forced swim test Mice were tested by placing in a glass cylinder (height Z 25 cm; diameter Z 10 cm) containing water (24–25  C) to a depth of 14 cm. The time that the animal

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spent trying to escape, immobile and moving around the tube (swimming) were recorded in 1-min time bins for 6 min. 2.8. Statistics Data were analysed by analysis of variance (ANOVA) followed by Dunnett’s t-test using BMDP statistical software package (BBN Products Corporation, USA). A p ! 0.05 was taken as significant. All data were presented as mean G s.e.m. unless otherwise stated. 2.9. Hypothalamic slices preparations Hypothalamic slices containing the SCN were obtained during the light phase (generally between ZT2–5) from male mice (3–4 months old). Tissue was maintained in a gas–fluid interface slice chamber at 34.5  C and continuously perfused with oxygenated ACSF. Drugs were warmed to 34.5  C and applied as a 200-nl microdrop to the SCN area on the first day in vitro. Antagonists were applied 5 min prior to agonist treatment. Extracellular recordings were made from spontaneously discharging neurones beginning from ZT0 on the second day in vitro. The average spontaneous firing rate (measured for 3 min) and the ZT for each single unit encountered was recorded by an experimenter blind to all treatments. Slices without significant differences across firing rate data grouped into 1 h bins ( p ! 0.05; ANOVA) were not used for further analysis. If there were significant differences, data were smoothed by 1 h running means with a 15-min lag. The time corresponding to the maximum of the smoothed data is used as the time of the peak firing. Phase shifts are measured relative to the average time of peak firing of control slices (treated with a microdrop of ACSF).

3. Results 3.1. Generation of 5-HT7 receptor knockout mice 5-HT7 receptor knockout mice were generated using a targeting vector to knockout exon 1 of the 5-HT7 gene (Fig. 1a). This vector was transfected into 129SvEv embryonic stem cells and six positive ES clones showing the targeted mutation were identified (Fig. 1b) and subsequently injected into C57Bl/6 blastocysts. A single independent line (line 3) was bred to generate homozygotic animals and all behavioural assessments have been carried out in male mice from the F3 or F4 generation of this line. PCR analysis of tail DNA confirmed the successful generation of homozygous knockout mice (Fig. 1c). Autoradiographic studies have confirmed that 5-HT7 receptors are absent in the knockout mice. Areas with high total [35S]-radiolabeled

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5-HT7 receptor antagonist (compound A) binding in wild type mice included septal, thalamic, hypothalamic and amygdaloid nuclei as expected. No specific binding in these areas was observed in the knockout mice, consistent with a lack of functional receptor protein. Representative sections demonstrating the loss of thalamic binding sites are shown in Fig. 1d. RT-PCR studies on 5-HT7 receptor knockout mouse brain RNA confirmed the loss of 5-HT7 receptor mRNA (data not shown). 3.2. Behavioural profile The 5-HT7 receptor knockout mice were first tested in neurological screens designed to reveal potential sensorimotor impairments that complicate interpretation of other behavioural data. The 5-HT7 receptor knockout mice grow and reproduce normally. The absence of major developmental abnormalities suggests that the receptor does not play an essential role during development. The mice also have normal body weight and basal rectal temperature compared with wild type mice, and appeared to be in good health. The mice showed normal balance and coordination when assessed on the rotarod and beam balance tests, indicating normal neurological function (Crawley and Paylor, 1997; Nakamura et al., 1999). Therefore, the mice were analysed in a number of assays sensitive to a range of psychoactive drugs. 3.3. Assays sensitive to potential psychotic and anxiety states The 5-HT7 receptor knockout mice exhibited the normal progressive increase in acoustic startle response with increasing decibel level, suggesting that the receptor may not play a significant role in this reflex (data not shown) and confirming the mice do not have impaired hearing. In the PPI paradigm, no difference was observed in the 5-HT7 receptor knockout mice compared to wild type controls (Fig. 2). Wild type and knockout mice were also assessed on the elevated plus maze. There was no difference in time spent exploring the open arms or number of entries onto the open arms of the maze (data not shown) between the two genotypes. 3.4. Assays sensitive to antidepressant drugs The 5-HT7 receptor knockout mice displayed a reduction in immobility compared with wild type controls in the Porsolt forced swim test (Fig. 3a). However, treatment of wild type mice with the 5-HT7 receptor antagonist, SB-258719 (at doses up to 30 mg/kg; i.p.), demonstrated only a small, non-significant decrease in immobility (Fig. 3b) whereas desipramine did produce

Fig. 2. Prepulse inhibition: (a) mean startle responses to 12 random repetitions of (i) recording alone (no stim), (ii–iii) a 20-ms, 80 dB or 90 dB prepulse alone, (pp80, pp90), (iv–vi) a 120-dB pulse preceded by a 70, 80, 90 dB prepulse (pp70p, pp80p, pp90p), (vii) a 40-ms, 120 dB pulse-alone were calculated for each individual mouse (pulse) and these were then used to determine the percentage prepulse inhibition of the acoustic startle (%PPI) for Htr7ÿ/ÿ mice (filled bars) and wild type mice (open bars). (b) Results are expressed as the mean %PPI G s.e.m. (n Z 12 mice/group) for Htr7ÿ/ÿ mice (filled bars) and wild type mice (open bars). %PPI Z (prepulse C pulse startle response (i.e. pp70p) divided by 120 dB pulse response) times 100.

a significant reduction in immobility in the same experiment. These assays were carried out during the light phase as is normal for behavioural testing. However, when mice were dosed with SB-258719 (30 mg/kg; i.p.) and tested in the dark phase, they did recapitulate the phenotype of the knockout mice (Fig. 3c), generating a similar reduction in total immobility to that observed following treatment with the antidepressant, desipramine, and in the knockout mice. There was no potentiation of immobility in the 5-HT7 receptor knockout mice when tested during the dark phase (Fig. 3c). A separate experiment comparing the effects of desipramine in the light and dark phase of the circadian rhythm also showed no potentiation when tested during the dark (data not shown).

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Fig. 3. Porsolt swim test: the time spent immobile for: (a) knockout (Htr7ÿ/ÿ) and wild type animals during light phase, (b) wild type animals treated with SB-258719 (30 mg/kg; i.p.) desipramine (DMI; 20 mg/kg; i.p.) or vehicle during light phase, (c) wild type or knockout (Htr7ÿ/ÿ) animals treated with SB-258719 or vehicle during dark phase. Data were recorded in 1-min time bins for 6 min. Mean data are expressed Gs.e.m. for n Z 16–18 mice/group. Data were subjected to ANOVA followed by Dunnett’s t-test. *p % 0.05 vs saline vehicle or wild type animals.

3.5. A role for 5-HT7 receptors in modulating circadian rhythms The hypothalamic suprachiasmatic nuclei function as an endogenous circadian pacemaker in mammals, and generate a self-sustained oscillation in vitro. This oscillation may be measured in the hypothalamic brain slice preparation as a rhythm in firing rate that can be observed for three to four days providing a unique system in which to examine the effect of drugs and other stimuli on the circadian clock. Hypothalamic slices containing the SCN were obtained from wild type and

knockout animals from male mice (3–4 months old) during the light phase. Extracellular recordings were made from spontaneously discharging neurones beginning from ZT0 on the second day in vitro. A typical phase shift to 8-OH-DPAT in wild type slices is shown in Fig. 4a. This phase shift is attenuated in the 5-HT7 KO mice (mean phase shift 1.6 G 0.2 h) compared to wild type controls (mean phase shift 3.2 G 0.2 h) shown as histograms in Fig. 4b. The amplitude of the firing rate rhythm in these animals was not significantly attenuated as compared with WT animals (data not shown). The residual phase shift to 8-OH-DPAT in the knockout

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independent line (line 3) was bred to generate homozygotic animals and all behavioural assessments have been carried out in male mice from the F3 or F4 generation of this line. PCR analysis of tail DNA confirmed the successful generation of homozygous knockout mice at the genome level (Fig. 1c) while RTPCR (data not shown) and autoradiographic studies (Fig. 1d) confirmed that functional 5-HT7 receptor mRNA and protein are absent in the knockout mice (Fig. 1d). A recent study demonstrated that the hypothermic effects of 5-HT are absent in 5-HT7 receptor knockout mice (Hedlund et al., 2003), a phenotype confirmed independently in our knockout mice (Guscott et al., 2003). A novel 5-HT7 receptor antagonist, SB-258719, was reported recently to be at least 100-fold selective against other 5-HT receptors and have good brain penetration following i.p. dosing in mice, suggesting it would be a useful in vivo tool (Forbes et al., 1998; Lovell et al., 2000). We previously demonstrated that SB-258719 dose dependently (5–20 mg/kg; i.p.) inhibited 5-CT induced hypothermia (Guscott et al., 2003). Therefore, we used doses of 10–30 mg/kg; i.p. in our subsequent behavioural experiments to ensure pharmacologically relevant blockade of 5-HT7 receptors. 4.2. Assays sensitive to antipsychotic and anxiolytic drugs

Fig. 4. Extracellular recording of firing rate rhythms. Panel a: peak times in hypothalamic slices from wild type mice treated with DPAT (200 nl microdrop, 10 mM, yellow trace) are advanced compared to untreated control slices. The lines indicate results from the running mean smoother for all cells recorded from untreated control slices (n Z 7 slices). Panel b is a histogram of phase shifts to 8-OH-DPAT alone, or in the presence of either pindolol (200 nl microdrop, 10 mM) or ritanserin (200 nl microdrop, 100 mM) in wild type and Htr7ÿ/ÿ mice (n Z at least 5 in each group).

mice was completely blocked by pindolol, acting as a 5-HT1A receptor antagonist, but not ritanserin. The phase shifts to 8-OH-DPAT in the WT mice were partly attenuated by both pindolol and ritanserin, indicating that actions at both the 5-HT7 and 5-HT1A receptors appear to contribute to the non-photic phase shifts to 8-OH-DPAT seen in the mouse.

4. Discussion 4.1. Characterisation of 5-HT7 receptor knockout mice and the selective 5-HT7 receptor antagonist, SB-258719 5-HT7 receptor knockout mice were generated by knocking out exon 1 of the 5-HT7 gene A single

Given the widespread limbic distribution of the receptor and the relatively high affinity of the receptor for drugs such as clozapine, a potential therapeutic utility in schizophrenia has been suggested. Schizophrenic patients and rats raised in social isolation show deficits in prepulse inhibition (PPI) that are thought to reflect a defect in sensorimotor gating (Geyer et al., 2001). The 5-HT7 receptor knockout mice exhibited the normal progressive increase in acoustic startle response with increasing decibel level, suggesting that the receptor may not play a significant role in this reflex (data not shown) and confirming the mice do not have impaired hearing. In the PPI paradigm, the response to an acoustic startle is reduced if that tone is preceded by another sub-threshold tone (prepulse) which itself does not elicit a startle response and is used in humans and animals to study sensorimotor gating mechanisms. Many antipsychotic drugs can reverse PPI. However, no difference in PPI was observed in the 5-HT7 receptor knockout mice compared to wild type controls (Fig. 2). Therefore, at least in this experimental paradigm there is no evidence for therapeutic utility of 5-HT7 receptor antagonists in the treatment of schizophrenia. Interestingly, it was recently demonstrated that SB-258741, a 5-HT7 receptor antagonist was able to normalise PCPdisrupted PPI (Pouzet et al., 2002) but not PCPdisrupted social interaction or D-amphetamine induced

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hyperactivity leading the authors to also conclude that 5-HT7 receptor antagonists are unlikely to be effective in schizophrenia. It would be of interest to compare these findings with the effects of PCP on behaviour in the 5-HT7 receptor knockout mice. Wild type and knockout mice were also assessed on the elevated plus maze, a test used to assess anxiety-like behaviours in rodents (Handley and Mithani, 1984). There was no difference in time spent exploring the open arms or number of entries onto the open arms of the maze (data not shown) between the two genotypes. Therefore, at least in this paradigm, the 5-HT7 receptor does not appear to be a potential target for therapeutic intervention in anxiety.

4.3. Assays sensitive to antidepressant drugs The 5-HT7 receptor knockout mice displayed a reduction in immobility compared with wild type controls in the Porsolt forced swim test (Fig. 3a). This is one of the most commonly used rodent screens for antidepressant drugs (Porsolt, 2000). The duration of immobility in this assay is reduced by most clinically used antidepressants, including atypical agents such as mianserin. However, although the reduction in immobility observed in the knockout mice was of a similar extent to that observed with optimal doses of clinically effective antidepressants, treatment of wild type mice with the 5-HT7 receptor antagonist, SB-258719 (at doses up to 30 mg/kg; i.p.), demonstrated only a small, non-significant decrease in immobility (Fig. 3b). Such doses of compound are predicted to be more than enough to fully block the 5-HT7 receptor in vivo. Parallel experiments using the antidepressant desipramine (20 mg/kg; i.p.) demonstrated significantly decreased immobility as expected (Fig. 3b), demonstrating that the absence of effect was not due to a lack of sensitivity in the assay. The observed mismatch between the phenotype of the knockout mouse and the effects of the antagonist in normal animals could be explained by: (1) the phenotype of the knockout mouse being due to adaptive changes rather than lack of receptors in the adult animals; (2) chronic dosing rather than acute dosing being required; (3) the Porsolt test conditions being suboptimal to detect 5-HT7 receptor antagonist activity. The knockout mice showed no difference in spontaneous locomotor activity compared to wild type controls (data not shown) and performed normally in various neurological tests suggesting this phenotype is a response to the experimental paradigm rather than a direct effect on locomotor activity. The need for chronic administration would be difficult to test as the pharmacokinetic properties of SB-258719 are not conducive to chronic dosing (plasma half-life in rodents !1 h, data not

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shown). Also, antidepressants routinely work in this test when dosed acutely. 5-HT7 receptors have been proposed to play a role in circadian rhythm biology, and most experiments on mice are carried out during the light phase. However, this may not be the best time to study mouse behaviour as they are nocturnal creatures and the light phase is their subjective night (Jones and King, 2001). Therefore, we tested SB-258719 (30 mg/kg; i.p.) in the forced swim test during the dark phase. Interestingly, under these conditions the 5-HT7 receptor antagonist did recapitulate the phenotype of the knockout mice (Fig. 3c), generating a similar reduction in total immobility to that observed following treatment with the positive controls and in the knockout mouse. The significance of the phase difference in effect in this behavioural assay is unclear. Many clinically effective antidepressants do not demonstrate this light phase dependency of efficacy. Indeed, the knockout mouse demonstrated reduced immobility in the light and dark phase. This may be due to increased endogenous tone on the 5-HT7 receptor system found during the active phase of the mouse’s day. Highest levels of serotonin release have been demonstrated to be associated with periods of high activity, which occur at night in nocturnal species (Dudley et al., 1998). Consistent with these findings, a recent study found SB-258741, a related 5-HT7 receptor antagonist, to be inactive in the mouse forced swim test when tested under normal conditions (Pouzet, 2002). Our study predicts that the compound may only be active if tested in the dark phase. Nonetheless, the experiments in this study provide preclinical support for the 5-HT7 receptor as a therapeutic target for depression and further link 5-HT7 receptor function to circadian biology.

4.4. A role for 5-HT7 receptors in modulating circadian rhythms The main circadian pacemaker mediating daily rhythmicity in mammals is located in the suprachiasmatic nucleus (SCN) (Rusak and Zucker, 1979). Environmental stimuli such as light can reset the pacemaker. The SCN receives photic input via a direct glutamatergic pathway to the retina and input via a serotonergic (5-HT) projection from the midbrain raphe nuclei. Modulations in 5-HT levels have been shown to reset the pacemaker and alter responsiveness of the system to light (Rea and Pickard, 2000; Smart and Biello, 2001). It appears that these effects of 5-HT are mediated by various receptor subtypes. Localisation studies demonstrated that 5-HT7 receptor mRNA and protein (To et al., 1995; Lovenberg et al., 1993; Neumaier et al., 2001) are expressed in the SCN.

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The 5-HT1A/7 receptor agonist, 8-hydroxy-2-(dipropylamino) tetralin (8-OH-DPAT), can dose-dependently attenuate phase-shifts of the SCN to light during the activity phase (Weber et al., 1998). Experiments, blocking these effects with pindolol or ritanserin or SB-269970, suggest that at least some of the effects of 8-OH-DPAT on circadian rhythms may be mediated by the 5-HT7 receptor subtype (Prosser et al., 1989; Kawahara et al., 1994; Thomas et al., 1998; Quintero and McMahon, 1999; Duncan et al., 2004). In mouse, 5-HT7 receptor immunoreactivity is associated with dendrites and axon terminals of GABA, vasopressin and VIP immunoreactive neurons, and these are contained within the rostrocaudal extent of the SCN (Belenky and Pickard, 2001). Further, optic nerveevoked potentials in mouse SCN neurons are inhibited by DPAT in the presence of ritanserin, but not WAY100635, suggesting a 5-HT7 receptor involvement (Smith et al., 2001). The hypothalamic suprachiasmatic nuclei function as an endogenous circadian pacemaker in mammals, and generate a self-sustained oscillation maintained in hypothalamic brain slice preparations for three to four days providing a unique system in which to examine the effect of drugs and other stimuli on the circadian clock. Hypothalamic slices containing the SCN were obtained from wild type and knockout animals and extracellular recordings made. The phase shift to 8-OH-DPAT in wild type slices (Fig. 4a) is attenuated in the 5-HT7 KO mice (Fig. 4b). The amplitude of the firing rate rhythm in these animals was also significantly attenuated as compared with WT animals (data not shown). The residual phase shift to 8-OH-DPAT in the knockout mice was completely blocked by pindolol, acting as a 5-HT1A receptor antagonist, but not ritanserin (acting as a 5-HT7 receptor antagonist) confirming directly that 5-HT7 receptors are involved. The phase shifts to 8-OHDPAT in the WT mice were partly attenuated by both pindolol and ritanserin, indicating that actions at both the 5-HT7 and 5-HT1A receptors appear to contribute to the non-photic phase shifts to 8-OH-DPAT seen in the mouse. This agrees with findings by Sprouse et al. (2003) in which pindolol blocked the in vitro phase shifting effect of 8-OH-DPAT in wild type and 5-HT7 receptor knockout mice, but this effect, and the phase shifting effects of 5-HT1A/2/7 agonists, has so far not been demonstrated in vivo (see Antle et al., 2003). This is also in agreement with in vivo effects found in hamster (Tominaga et al., 1992). Interestingly, a disturbed circadian rhythm is a core symptom of clinical depression (Kupfer, 1995; Wehr et al., 1979). It is tempting to speculate that the possible role of 5-HT7 receptors in the modulation of circadian rhythms may add to the potential for 5-HT7 receptor antagonists to treat depression. Interestingly, a recent report (Thomas et al., 2003) suggested that 5-HT7 receptor antagonists reduce

paradoxical (REM) sleep in rats perhaps by circadian modulation and implied clinical utility in the treatment of sleep disorders associated with depression. In summary, the 5-HT7 receptor knockout mouse displays an ‘‘antidepressant-like’’ phenotype in the forced swim test, a preclinical assay routinely used to assess antidepressant potential. The 5-HT7 receptor antagonist, SB-258719, also generated robust antidepressant-like effects in this assay, but only when tested in the animal’s dark, or active, phase confirming that the phenotype is due to the lack of an active receptor rather than the consequence of developmental adaptation. The hypothalamic slice data confirm directly that 5-HT7 receptors mediate the phase shifting properties of 8-OHDPAT and suggest a role in regulating circadian rhythms. These data strongly support the further investigation of 5-HT7 receptor antagonists for the treatment of depression, and suggest potential in the circadian rhythm/sleep disturbance area.

Acknowledgments We would like to thank Dennis Dean and colleagues in the Labeled Compound Synthesis Group (LCS) at MRL Basic Research, Rahway for assistance in the generation of (R)-[35S]-1-[2-(1-benzenesulfonyl-pyrrolidin-2-yl)-ethyl]-4-methyl-piperidine.

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