Introduction

Top Summary Introduction Experimental Procedures Results Discussion References

The neurotransmitter 5-hydroxytryptamine (5-HT) is involved in many physiological functions (Hoyer et al., 1994). In mammals, the multiple actions of 5-HT are mediated by the interaction with at least 13 molecularly distinct receptors (Saudou and Hen, 1994). Among them, the 5-HT_1B receptor has gained particular interest. It belongs to the superfamily of G protein-coupled receptors, and its principal signal transduction pathway occurs via negative coupling to adenylyl cyclase (Adham et al., 1992; Hamblin et al., 1992; Maroteaux et al., 1992; Weinshank et al., 1992). The 5-HT_1B receptors modulate serotonergic transmission by a presynaptic autoreceptor function (Middlemiss et al., 1988; Hamblin et al., 1992). They also appear to function as presynaptic heteroreceptors and have been shown to regulate the release of norepinephrine, glutamate, acetylcholine, and -aminobutyric acid (Raiteri et al., 1986; Hoyer and Middlemiss, 1989; Molderings et al., 1990; Hen, 1992). Furthermore, they may also play a role in the presynaptic inhibition of neuropeptide release (substance P and calcitonin gene-related peptide) (Buzzi et al., 1991; Moskowitz, 1992; Bonaventure et al., 1998a). The 5-HT_1B receptors are widely distributed in the brain but are particularly abundant on axon terminals in the output structures of the basal ganglia (globus pallidus and substantia nigra) (Waeber et al., 1989; Bruinvels et al., 1993; Boschert et al., 1994; Langlois et al., 1995; Bonaventure et al., 1997, 1998b). Because of their role in the regulation of multiple neurotransmitters and neuropeptides, 5-HT_1B receptors could be a target for the development of new therapeutic agents in the field of depression and migraine, among others (Halazy et al., 1997).

Despite an amino acid sequence identity of more than 90% with the human (h)5-HT_1B receptor, the rodent 5-HT_1B receptors display different ligand binding properties than their human homolog. For example, the antimigraine agent alniditan binds with a 50-fold higher affinity to the human compared with the rodent 5-HT_1B receptor, whereas CP93129 and certain adrenergic antagonists bind more potently to the rodent receptor (Hamblin et al., 1992; Bach et al., 1993; Leysen et al., 1996; Zgombick et al., 1997). These differences in pharmacological properties result from an amino acid substitution at position 355, being asparagine in rat and mouse and threonine in the human receptor (Metcalf et al., 1992; Oksenberg et al., 1992; Parker et al., 1993).

As a consequence of this species difference, rats and mice are not suitable or representative for testing the pharmacological properties of h5-HT_1B receptor agonists and antagonists. Transgenic animal technology has provided tools to delete or replace genes that encode receptors; hence, genes can be "humanized," and the function of the human protein can be studied in the transgenic animal. Mutant mice lacking a functional gene encoding the 5-HT_1B receptor have been generated by homologous recombination, resulting in mice with increased aggressive behavior (Saudou et al., 1994).

In the present study, an animal model to investigate the h5-HT_1B receptor was generated using a "knock-in" technology (i.e., by "humanizing" the mouse locus), producing mice that express the h5-HT_1B receptor instead of its murine counterpart but from the same genetic locus. We report the characterization and functional authentication of the h5-HT_1B receptor as expressed in the brain of homozygous transgenic animals. The distribution patterns of h5-HT_1B and murine (m)5-HT_1B receptor mRNA and binding sites in transgenic and wild-type mice were compared by quantitative in situ hybridization histochemistry (ISHH) and receptor autoradiography throughout the brain with two different radioligands (i.e., the antagonist [³H]GR125743, which recognizes both m5-HT_1B and h5-HT_1B receptors [Mengod et al., 1996; Audinot et al., 1997; Domenech et al., 1997] and the agonist [³H]alniditan, which is selective for h5-HT_1B receptors [Leysen et al., 1996]. Additionally, [³H]GR125743 concentration binding curves were obtained from brain sections containing the substantia nigra and globus pallidus of wild-type and transgenic animals. Differential identification was performed by measuring the potency of several compounds [alniditan, rauwolscine, 8-hydroxy-2-dipropylaminotetraline (8-OH-DPAT), CP93129, RU24969, pindolol, propranolol, sumatriptan, and 5-HT) to inhibit binding of [³H]GR125743 in transgenic and wild-type mice and in human brain sections by quantitative receptor autoradiography. In transgenic mouse brain, the functional coupling of the h5-HT_1B receptor to G protein activation was established by quantitative autoradiography of [³⁵S]guanosine-5'-O-(3-thio)triphosphate (GTPS) binding after stimulation by nonselective agonists [5-carboxamidotryptamine (5-CT) and 5-HT] or agonists selective for the mouse (CP93129) or human (alniditan) 5-HT_1B receptors. Modulation of electrically evoked [³H]5-HT release by a selective 5-HT_1B antagonist (SB224389) was studied in slices of transgenic mouse cortex, hippocampus, and striatum. In vivo effects (i.e., RU24969-induced locomotor activity and effects on body temperature) that are reported to be modulated by 5-HT_1B receptor activation (Ramboz et al., 1996; Hagan et al., 1997) were also investigated in these "humanized" mice.

	Experimental Procedures

Top Summary Introduction Experimental Procedures Results Discussion References

Materials. The mouse strain genomic library was obtained from Genome Systems (St. Louis, MO). The pSG5 expression vector, pBS (KS) vector, and Pful DNA polymerase were purchased from Stratagene (La Jolla, CA). Trizol reagent was from GIBCO BRL (Merelbeke, Belgium).

Generation of h5-HT_1B Receptor Transgenic Mice. The replacement or knock-in construct was based on the h5-HT_1B receptor gene (Jin et al., 1992), cloned in a pUC18 vector and on a genomic BAC clone containing the homologous mouse gene isolated from a mouse 129 strain genomic library (clone address BAC-231-B2). Both genes are intronless and thus easily manipulated. A 1173-bp EcoRI/BamHI fragment containing the h5-HT_1B receptor gene was subcloned in the pSG5 expression vector and amplified with adaptor primers containing suitable restriction sites (NcoI, underlined): forward primer 5'-ATAGCTAGCAGGCCTGCCACCATGGAGGAACCGGGTGCTCAG-3' including the ATG translation start codon of the human receptor gene (reverse complement, CAT, in bold) and reverse primer 5'-CCAGCCATGGTAAGATACATTGATGAGTTTGGACA-3', located 3' of the polyadenylation signal in pSG5. A 1.4-kb NcoI fragment encoding the human receptor gene with the SV40 polyadenylation signal of the vector was isolated.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Transgenic targeting construct, genotyping of ES cells, and analysis of mRNA expression. A, recombinant DNA construct used to target the m5-HT1B receptor gene and replace it with the h5-HT1B receptor gene. The black box represents the coding sequence of the intronless h5-HT1B receptor gene; the hatched box represents the HYG marker gene, which was embedded in the SauI restriction site (S); and the small box with bricks represents the SV40 polyadenylation signal. The unique SalI restriction site used to linearize the construct is also indicated. The wild-type m5-HT1B receptor gene (intronless) containing the coding sequence is represented by the gray box. Relevant restriction sites are shown: KpnI (K), EcoRV (E), and SauI (S). R (right) and L (left) represent the position of the genomic DNA probes, located externally to the genomic region that was targeted and used in the construct (white box). The predicted structure of the targeted 5-HT receptor gene contains the elements from the targeting vector (described above) and predicts the loss of the wild-type 3.4- and 7-kb KpnI fragments and of the 4.4-kb EcoRV fragment, while generating a 11.2-kb KpnI fragment and a 3.9-kb EcoRV fragment (, all boundaries). , position of the primers used for PCR amplification (sequences given in Experimental Procedures). B, example of Southern blot of DNA isolated from one of the four selected ES cell lines, digested with KpnI or EcoRV as indicated, and hybridized with the different probes (R, L, and HYG). See text and A for details. The leftmost lane (M) represents radiolabeled DNA markers with the approximate size indicated. C, Northern blot of total RNA isolated from the cerebrum, cerebellum, and liver of wild-type mice (lanes 1), heterozygous targeted mice (lanes 2), and homozygous targeted, humanized 5-HT1B mice (lanes 3). The blot was hybridized with the m5-HT1B receptor probe (top), the h5-HT1B receptor probe (middle), and a -actin cDNA probe (bottom).

RNA Extraction and Northern Blotting. Total RNA was extracted and purified from cerebrum, cerebellum, spleen, and liver of wild-type, heterozygous, and homozygous mice. The isolated organs were immediately frozen in liquid nitrogen and stored at 70°C until RNA extractions were performed with Trizol reagent. Tissue (100 mg) was mechanically homogenized (Virtis) in 1 ml of reagent and centrifuged at 16,000g for 15 min at 4°C. Chloroform (0.2 ml) was added to the supernatant, shaken vigorously, and left at ambient temperature for 3 min. After centrifugation at 16,000g for 10 min at 4°C, the aqueous phase containing RNA was mixed with 0.5 ml of isopropyl alcohol. After 15 min at ambient temperature, the RNA was pelleted at 16,000g for 10 min at 4°C. The RNA pellet was washed in 1 ml of 75% ethanol, resuspended in deionized formamide, stored at 70°C. The concentration of RNA in the samples was measured spectrophotometrically at 260 nm. Then, 10 µg of total RNA was separated and blotted as described previously (Lorent et al., 1994). The h5-HT_1B receptor mRNA transcript was detected with a probe that represented the entire 1.3-kb human cDNA. The m5-HT_1B receptor probe was a 0.7-kb fragment generated by PCR with forward primer 5'-CATTTACCAGGACTCCATCGC-3' (position 651 in the mouse cDNA) and reverse primer 5'-GAGACTCGCACTTTGACTTGG-3' (position 1421 in the mouse cDNA) (Maroteaux et al., 1992). Equal loading of mRNA was authenticated by hybridization with a 2-kb -actin cDNA probe (Umans et al., 1995).

Tissue Preparation for ISHH and Radioligand Autoradiography. Transgenic or wild-type mice (20-35 g) were decapitated. Brains were immediately removed from the skull and rapidly frozen in dry-ice-cooled 2-methylbutane (40°C). Then, 20-µm-thick frontal sections were cut using a Reichert Jung 2800E cryostat-microtome (Cambridge Instruments, Cambridge, UK) and thaw-mounted on adhesive microscope slides (Super Frost). The sections were kept at 70°C until use. The sectioning protocol for the regional distribution study included series of coronal sections covering the entire brain (n = 3-6) with an interspace of 300 µm. Consecutive sections were used for ISHH, receptor autoradiography with [³H]GR125743 and [³H]alniditan. One additional brain was used for horizontal sections; six additional brains were sectioned in the coronal plane at the level of globus pallidus and substantia nigra and used to generate inhibition curves and concentration binding curves (see below) and for autoradiography of agonist-stimulated [³⁵S]GTPS binding.

Quantitative ISHH. ISHH was performed with [³⁵S]UTP-labeled cRNA probes as described previously (Bonaventure et al., 1998b). A segment of DNA encoding the h5-HT_1B receptor (full length; Leysen et al., 1996) was subcloned into pRcCMV. Riboprobes were produced using a SP6 (antisense) or T7 (sense) transcription system in a standard labeling reaction mixture (Bonaventure et al., 1998b). Brain sections were thawed and fixed in paraformaldehyde, acetylated, dehydrated, and delipidated. The fixed sections were hybridized with 1 × 10⁶ cpm [³⁵S]UTP-labeled riboprobe per section. The diluted probe was applied to sections and hybridized at 50°C overnight in a humid chamber. After stringency washes, sections were exposed to Hyperfilm-³H for 4 weeks. ¹⁴C-standard strips previously cross-calibrated to ³⁵S were coexposed to allow densitometry. Films were developed manually in Kodak D-19 and fixed with Kodak Readymatic. The following control experiments were performed to determine the specificity of the hybridization signal: ISHH with sense probe and RNase treatment before hybridization with antisense probe.

Receptor Autoradiography. Sections were thawed and dried under a cold air stream and then preincubated three times for 5 min in 50 mM Tris·HCl buffer, pH 7.4, at an ambient temperature by immersing the sections on the microscope slides into a 400-ml jar. Next, they were incubated (drop incubation, 150 µl placed on each section) for 60 min at an ambient temperature in medium containing 4 nM [³H]alniditan or 2 nM [³H]GR125743, 50 mM Tris·HCl buffer, pH 7.4, 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 0.1% BSA, 0.01% ascorbic acid, and 2 µM pargyline (Bonaventure et al., 1998b). To measure [³H]alniditan binding, 100 nM 8-OH-DPAT was added to occlude the 5-HT_1A receptors. For concentration binding curves, [³H]GR125743 was used at 0.1, 0.5, 1, 2, 4, 8, and 10 nM. Inhibition of [³H]GR125743 binding by alniditan, 8-OH-DPAT, rauwolscine, CP93129, RU24969, pindolol, propranolol, sumatriptan, and 5-HT (using 15 concentrations per compound, within a range of 10¹¹ to 10⁴ M) was performed on mouse brain sections at the level of the globus pallidus and substantia nigra and on human substantia nigra sections. Nonspecific binding was measured in the presence of 10 µM 5-HT. After incubation, the excess of radioligand was washed off by immersing the microscope slides in a jar (five times for 1 min) with Tris·HCl buffer, pH 7.4, at 4°C followed by a quick rinse in water and drying under a cold air stream. The sections and standard tritiated plastic microscales were placed in a light-tight cassette and covered with a light-sensitive Hyperfilm-³H. After 6 weeks' exposure, they were developed manually in Kodak D-19 developer for 2 min and fixed with Kodak Readymatic for 3 min.

Quantitative Autoradiography of Agonist-Stimulated [³⁵S]GTPS Binding. [³⁵S]GTPS binding was visualized using the method described by Waeber and Moskowitz (1997) with slight modifications. Briefly, the tissue sections, brought to an ambient temperature 15 min before the experiments, were incubated for 30 min at an ambient temperature in 50 mM HEPES buffer, pH 7.5, containing 100 mM NaCl, 3 mM MgCl₂, 0.2 mM EGTA, 0.01% BSA, and 0.2 mM dithiothreitol; they were incubated for an additional 15 min in the same fresh buffer supplemented with 2 mM GDP. Agonist-stimulated binding was measured by incubating the sections for 60 min at 30°C in buffer containing 2 mM GDP, 0.04 nM [³⁵S]GTPS, and 10 µM agonist (alniditan, CP93129, 5-HT, or 5-CT). Basal activity was determined in the absence of agonist. Nonspecific binding was assessed by including 10 µM unlabeled GTPS in the incubation buffer. Slices were washed twice for 3 min in ice-cold 50 mM HEPES buffer, pH 7.0, dipped briefly in ice-cold distilled water, dried under a cold air stream, and exposed to Hyperfilm-max for 72 h. ¹⁴C standards strips previously cross-calibrated to ³⁵S standards were coexposed to allow densitometry. Films were developed manually in Kodak D-19 (2 min) and fixed with Kodak Readymatic (3 min).

Data Analysis of ISHH, Receptor Autoradiography, and Agonist-Stimulated [³⁵S]GTPS Binding. Autoradiograms of ISHH, radioligand, or [³⁵S]GTPS binding were analyzed and quantified using an MCID-M4 3.0 image analysis system (Imaging Research, St-Catharines, Ontario, Canada). Optical densities in the anatomical regions of interest were transformed into levels of bound radioactivity after calibration of the image analyzer with gray values generated by the coexposed standards. The hybridization signal was expressed as dpm/mg tissue.

In Vitro Electrically Evoked [³H]5-HT Release Experiments. To save mice, the animals used for the in vitro release studies had been used previously in behavioral tests. Before sacrifice, both transgenic and wild-type mice had been drug free for at least 21 days.

In Vivo Functional Studies: Locomotor Activity and Hypothermia. To reduce the number of animals used, we resorted to a protocol in which the same animals received increasing doses of drugs.

Statistical Analysis. The results are presented as mean ± S.D. Wilcoxon matched pairs signed-ranks test (two-tailed) was used for comparing treated versus vehicle groups. The Mann-Whitney U test (two-tailed) was used for comparison of wild-type with transgenic animals and for comparing antagonist-treated versus vehicle group.

	Results

Top Summary Introduction Experimental Procedures Results Discussion References

Targeting of m5-HT_1B Receptor Gene by h5-HT_1B Receptor Gene

In the targeting construct, the mouse coding sequence was replaced by the human under the control of the mouse promoter and a necessary positive selection marker was placed downstream of the gene in a SauI site, in antisense orientation to minimally affect expression of the 5-HT_1B gene (Fig. 1A). The replacement vector was linearized at the unique SalI site and electroporated into ES cells. Cells surviving electroporation were grown in selective medium containing HYG, resulting in several thousands of colonies, of which 342 were isolated, expanded, and analyzed by genomic Southern blotting.

The first genotyping of these 342 colonies was by Southern blotting after restriction with KpnI and hybridization with the L probe to reveal fragments of 3.4 kb from the wild-type and 11.2 kb from the targeted allele (Fig. 1, A and B). By this criterion, 18 targeted ES cell lines were retained and expanded. DNA from these 18 lines was then analyzed with all three probes (L, R, and HYG, Fig. 1), yielding four ES cell lines with the expected KpnI restriction patterns. These lines were further authenticated by restriction with EcoRV and confirmed to contain a single copy of the PGK-HYG cassette (Fig. 1).

The four correctly targeted ES cell lines, representing an overall recombination frequency of 1.2%, were injected into C57Bl blastocysts and all resulted in coat color chimeric mice that transmitted the targeted gene through the germline. A total of 172 pups with brown coat color were analyzed by Southern blotting of tail-tip DNA, identifying 48 female and 43 male heterozygous mice (total of 53%) that were mated and used to establish homozygous offspring. In total, 154 pups resulting from matings of heterozygous animals were genotyped by Southern blotting of tail-tip DNA after restriction with EcoRV. This identified 46 wild-type mice (29.9%), 72 heterozygous mice (46.8%), and 36 homozygous targeted mice (23.3%), clearly establishing a normal Mendelian inheritance pattern.

Expression of 5-HT_1B Receptor mRNA

Total RNA isolated from cerebrum, cerebellum, spleen, and liver was analyzed by Northern blotting (Fig. 1C). The h5-HT_1B receptor cDNA probe revealed transcripts of about 2 to 2.4 kb in the cerebrum and cerebellum of heterozygous and homozygous targeted mice. The difference in size from those normally expressed in human tissue as described (Jin et al., 1992) results from the use of a different 3'-UTR in our construct, incorporating the SV40 poly(A)⁺ signal from the pSG5 vector (see Experimental Procedures). No equivalent h5-HT_1B receptor transcripts were detected in wild-type mice. Subsequent hybridization of the same blots with the m5-HT_1B receptor cDNA probe revealed a 6-kb transcript as expected (Maroteaux et al., 1992) in wild-type and in heterozygous mice, whereas no transcript was detected in the homozygous h5-HT_1B receptor recombinant mice. No transcripts were detected in the liver of these same animals with either probe (Fig. 1C).

Quantitative ISHH

Control experiments were carried out to determine the specificity of the hybridization. Incubation with sense probe or RNase pretreatment before hybridization with antisense probe did not yield a hybridization signal (result not shown). The [³⁵S]cRNA probe generated by in vitro transcription from full-length cDNA coding for the h5-HT_1B receptor has been used for hybridization on transgenic and wild-type mouse brain sections. The high percentage of sequence identity (88%) between the h5-HT_1B and m5-HT_1B receptor gene allows a direct comparison using the same ³⁵S-labeled riboprobes between h5-HT_1B and m5-HT_1B receptor mRNA levels of expression in transgenic and wild-type mice, respectively.

The distribution patterns of h5-HT_1B and m5-HT_1B receptor mRNA in homozygous transgenic and wild-type mice, respectively, were identical (Table 1, Fig. 2). Also, the h5-HT_1B and m5-HT_1B receptor mRNA densities measured in transgenic and wild-type animals were comparable (Table 1).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Comparison of the anatomic distribution of m5-HT1B and h5-HT1B receptor mRNA in autoradiograms of a horizontal section of the wild-type (A) and transgenic (B) mouse brain, respectively. OT, olfactory tubercle; CPu, caudate putamen; Hip, hippocampal formation; DR, dorsal raphe nucleus; Cer, cerebellum. Note that the distribution patterns and densities are completely similar in transgenic and wild-type mouse brain. Scale bar, 5 mm.

The main sites of 5-HT_1B receptor mRNA expression in both wild-type and transgenic mice were the caudate-putamen, the CA1 field of the hippocampal formation, and the Purkinje cell layer of the cerebellum (Fig. 2, Table 1). High 5-HT_1B receptor mRNA levels could also be detected in the dorsal raphe nucleus (Table 1), nucleus accumbens, and olfactory tubercle (not quantified). Moderate expression was found in the lateral geniculate nucleus of the thalamus (Table 1) and throughout the cortical mantle (not quantified). No significant levels of 5-HT_1B receptor mRNA were detected within the globus pallidus and the substantia nigra of transgenic and wild-type animals.

Anatomic Distribution of [³H]GR125743 and [³H]Alniditan Binding Sites in Transgenic and Wild-Type Mouse Brain

The distribution pattern of 5-HT_1B binding sites in transgenic and wild-type mouse brain was compared by radioligand binding autoradiography in frontal sections. Two different radioligands were used: the antagonist [³H]GR125743, recognizing m5-HT_1B and h5-HT_1B receptors, and the agonist [³H]alniditan, recognizing h5-HT_1B receptors selectively under the conditions used. Illustrations of receptor labeling in the ventral pallidum of wild-type and transgenic mouse are shown in Fig. 3. A list of quantified [³H]GR125743 (2 nM) and [³H]alniditan (4 nM) binding sites in wild-type and transgenic mouse brain areas is presented in Table 1.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   [3H]GR125743 (2 nM) (A-D) and [3H]alniditan (4 nM) (E-H) binding in ventral pallidum of transgenic (A, B, E, and F) and wild-type mice (C, D, G, and H) visualized by receptor autoradiography (frontal sections). B, D, F, and H, nonspecific binding in the presence of 10 µM 5-HT. VP, ventral pallidum. Note the absence of [3H]alniditan labeling in wild-type mouse ventral pallidum (G, open arrow). Scale bar, 5 mm.

The pattern of [³H]GR125743 binding sites throughout the transgenic and wild-type mouse brain was completely similar (Table 1, Fig. 3). Consistently, higher [³H]GR125743 binding densities were measured in wild-type mouse brain, which is confirmed by the ligand concentration binding curves (see below). In both wild-type and transgenic animals, the highest densities were found in the two output structures of the basal ganglia (substantia nigra and globus pallidus). Medium-dense labeling was observed in the superficial layer of the superior colliculus, dorsal subiculum, CA1 field of the hippocampal formation, and central gray. Weak binding was found in the caudate-putamen, nucleus accumbens, interpeduncular nucleus, and lateral geniculate nucleus of the thalamus.

With [³H]alniditan, the same distribution pattern as obtained with [³H]GR125743 was found in transgenic mouse brain, but no specific labeling was detected in wild-type mice (Table 1, Fig. 3). The labeling intensity of [³H]alniditan in transgenic mice was lower than the intensity measured with [³H]GR125743 (Table 1).

Ligand Concentration Binding Curves

To compare the receptor density of the h5-HT_1B receptor expressed in transgenic mice and the m5-HT_1B receptor in wild-type mice, concentration binding curves of [³H]GR125743 were performed on brain sections at two levels: the substantia nigra and the globus pallidus.

Transgenic and wild-type mice of the same age (10 weeks) were used in this study.

Derived B_max and K_D values are listed in Table 2. The nonspecific binding determined in the presence of 10 µM 5-HT was linear. The affinity of [³H]GR125743 was comparable in wild-type and transgenic animals based on the overlap between the 95% confidence interval of K_D values (Table 2). The B_max values were higher in the substantia nigra than in the globus pallidus (Table 2). In both regions, higher B_max values were found in wild-type mice. The level of [³H]GR125743 binding sites measured in transgenic mice reached approximately 70% of [³H]GR125743 binding sites in wild-type mice.

Inhibition of [³H]GR125743 Binding in Transgenic Mice, Wild-Type Mice, and Human Brain Sections

Further pharmacological characterization was performed by measuring the potency of several compounds (alniditan, rauwolscine, 8-OH-DPAT, CP93129, RU24969, pindolol, propranolol, sumatriptan, and 5-HT) to inhibit specific [³H]GR125743 binding in wild-type and transgenic mouse and in human brain sections using quantitative autoradiography.

Inhibition curves were generated from measurements in substantia nigra and globus pallidus. The pIC₅₀ values are listed in Table 3.

Alniditan, rauwolscine, and 8-OH-DPAT displayed a higher affinity for the h5-HT_1B receptor than for the m5-HT_1B receptor (Table 3). CP93129, RU24969, pindolol, and propranolol showed a higher affinity for the mouse receptor (Table 3). The absence of inhibition of [³H]GR125743 binding in the transgenic mouse substantia nigra by CP93129 is illustrated in Fig. 4. The affinities of sumatriptan and 5-HT were similar for the transgenic and wild-type mouse receptors (Table 3). The pIC₅₀ values derived from the inhibition curves in transgenic mouse and human substantia nigra were very similar; a highly significant correlation between pIC₅₀ values in both tissues was obtained (Table 3; Fig. 5A; r Pearson = .98; p < .0001). In contrast, no significant correlation was found between the binding affinities of the compounds for the m5-HT_1B receptor in wild-type animals and the h5-HT_1B receptor expressed in transgenic animals (r = .18, p > .05, Fig. 5B) or the h5-HT_1B receptor in human substantia nigra (r = .05, p > .05, Fig. 5C).

View larger version (65K): [in this window] [in a new window]   Fig. 4.   Inhibition of [3H]GR125743 binding by CP93129 (3 µM) in wild-type (A-C) or transgenic (D-F) mice and in human substantia nigra (G-I) visualized by receptor autoradiography (frontal sections). A, D, and G, total binding. B, E, and H, binding in the presence of 3 µM CP93129. C, F, and I, nonspecific binding in the presence of 10 µM 5-HT. SN, substantia nigra; Sub, subiculum. Note the absence of inhibition of [3H]GR125743 in the transgenic mouse substantia nigra (B, open arrow) and in human brain. Scale bar, 5 mm.

View larger version (9K): [in this window] [in a new window]   Fig. 5.   Correlation between pIC50 values of compounds (see Table 3) for (A) the 5-HT1B receptor in human substantia nigra and the 5-HT1B receptor in transgenic mouse substantia nigra, (B) the 5-HT1B receptor in wild-type mouse substantia nigra and the 5-HT1B receptor expressed in transgenic mouse substantia nigra, and (C) the 5-HT1B receptor from wild-type mouse substantia nigra and the 5-HT1B receptor from human substantia nigra. The correlation coefficient (r Pearson) is listed in each panel. Note the high correlation coefficient between the pIC50 values for the h5-HT1B receptor expressed in transgenic mouse and the human substantia nigra.

Quantitative Autoradiography of Agonist (5-HT, 5-CT, CP93129, and Alniditan)-Stimulated [³⁵S]GTPS Binding in Transgenic and Wild-Type Mouse Brain Sections

The functional coupling of the h5-HT_1B receptor expressed in transgenic animals to G protein activation was investigated and compared with that of the mouse receptor by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPS binding. As previously reported (Waeber and Moskowitz, 1997), optimal agonist stimulation was observed in the presence of 10 µM agonist and 2 mM GDP at 30°C. Basal activity was determined in the absence of agonist and nonspecific binding in the presence of 10 µM GTPS. The specificity of the agonist-induced signal was demonstrated by inhibiting the labeling with a 5-HT_1B receptor antagonist (GR127935, results not shown). Percentages of agonist-induced stimulation of [³⁵S]GTPS binding are reported in Table 4; autoradiograms are shown in Fig. 6. Both 5-HT and 5-CT stimulated [³⁵S]GTPS binding in the substantia nigra and globus pallidus of wild-type and transgenic animals, with the highest stimulation in substantia nigra. The percentage of stimulation measured in the presence of 5-CT was higher than that in the presence of 5-HT (Table 4) in both wild-type and transgenic mice. For both agonists (i.e., 5-HT and 5-CT), the percentage of stimulation observed in wild-type mice was higher than that in transgenic animals. The selective rodent 5-HT_1B receptor agonist CP93129 was effective in wild-type but not in transgenic mice (Table 4, Fig. 6, E and F). Conversely, alniditan (a selective h5-HT_1B agonist) stimulated [³⁵S]GTPS binding in transgenic but not in wild-type animals (Table 4, Fig. 6, G and H).

View this table: [in this window] [in a new window]   TABLE 4 Quantitative autoradiography of agonist-stimulated [35S]GTPS binding in transgenic and wild-type mouse brain Agonist-stimulated [35S]GTPS binding was expressed as a percentage of basal activity (n = 3; mean ± S.D.).

View larger version (48K): [in this window] [in a new window]   Fig. 6.   Effect of agonists on [35S]GTPS binding in wild-type (A, C, E, G, and I) or transgenic (B, D, F, H, and J) mouse substantia nigra visualized by autoradiography (frontal sections). A and B, stimulated by 10 µM 5-HT. C and D, stimulated by 10 µM 5-CT. E and F, stimulated by 10 µM CP93129. G and H, stimulated by 10 µM alniditan. I and J, basal activity. SN, substantia nigra. Note the absence of stimulation of CP93129 in transgenic mouse substantia nigra and the stimulation of [35S]GTPS binding by alniditan in transgenic mouse substantia nigra (open arrow, F and H). Scale bar, 5 mm.

Modulation of [³H]5-HT Release: Effects on Basal and Electrically Evoked Release of Mouse Strain and Pharmacological Modulation

Basal and electrically induced overflow of [³H]5-HT from cerebral cortex, hippocampal, and striatal slices were studied in both wild-type and transgenic mice to evaluate the functional activity of the 5-HT_1B receptor in the two strains. The effect on 5-HT_1B receptors by exogenously applied agonist or antagonist was measured on basal and electrically stimulated ³H overflow. An appropriate agonist was used for the strains: 1 µM RU24969 for the wild-type mice and 1 µM alniditan for the transgenic mice. The applied concentrations were 30-fold above their pIC₅₀ values to inhibit [³H]GR125743 binding to 5-HT_1B receptors in brain sections of wild-type and transgenic mice.

Electrically stimulated ³H release in the absence of added drugs was lower in the hippocampus of wild-type than in that of transgenic mice (Table 5, p < .0001, Student's t test); in the other brain areas, no difference was seen between the two strains. Also, the evoked release compared with basal was significantly bigger in transgenic than in wild-type mouse hippocampus (Table 5, p < .0001, Student's t test). In none of the investigated brain areas of either mouse strain was a significant difference in the control S2/S1 ratios observed, measured in the absence of added drugs.

Neither agonist had a statistically significant effect on basal (fraction 12/fraction 2 ratio) or induced release (S2/S1 ratio) (Fig. 7). The use of alniditan (1 µM) also failed to affect [³H]5-HT overflow in the three brain areas studied of the wild-type mice (data not shown). The m5-HT_1B and h5-HT_1B receptor antagonist SB224289, however, was able to significantly increase the S2/S1 ratio in wild-type cerebral cortex and hippocampus but not in striatum. In cerebral cortex of wild-type mice, SB224289 had a tendency to increase basal release (fraction 12/fraction 2 = 113 ± 2.1% in control and 121 ± 4.2% in the presence of SB224289), but this effect on basal release did not reach statistical significance. In the transgenic mice, SB224289 did not affect basal or induced [³H]5-HT overflow in any of the brain areas investigated (Fig. 7).

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Drug effects on electrically evoked [3H]5-HT release from cerebral cortex, hippocampus, and striatum of wild-type and transgenic mice. Superfusion with 1 µM concentration of the drugs started 25 min (SB224289) and 21 min (RU24969) before S2; controls were simultaneously superfused with Krebs' buffer only. All values are mean ± S.D. of four to seven experiments. The difference between drug treated and controls was statistically significant: *p < .05, **p < .01, Student's t test.

In Vivo Functional Studies: Locomotor Activity and Hypothermia

The in vivo effect of a 5-HT_1B agonist was investigated in both the transgenic and the wild-type mice on two parameters, locomotor activity and hypothermia, which are reported to be modulated by 5-HT_1B receptor activation (Ramboz et al., 1996; Hagan et al., 1997). The 5-HT_1B agonist RU24969 was used because of its ability to penetrate the blood-brain barrier.

Locomotor Activity. In animals administered a single injection of vehicle, locomotor activity after 30 min was comparable in wild-type and transgenic mice (p > .05, two-tailed Mann-Whitney U test; Fig. 8). No statistically significant difference was observed in habituation (seen as a decrease in locomotor activity on repeated testing with vehicle injections) between transgenic and wild-type mice (Fig. 8, p > .05, two-tailed Mann-Whitney U test). RU24969 caused a dose-related increase in locomotor activity in wild-type mice that reached significance from 0.63 mg/kg on (p < .05, two-tailed Wilcoxon PPSR test; Fig. 8A) and consisted of distinctive thigmotactic circling. No increase in locomotor activity or thigmotactic circling was observed in the transgenic mice (Fig. 8B).

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Locomotor activity of wild-type (A) and transgenic (B) mice after s.c. injection of increasing doses of RU24969 (solid columns) or vehicle (open columns). The bars represent 5-min cumulative locomotor activity counts (crossings, mean ± S.D.; n = 5). Note the dose-related increase in locomotor activity in wild-type mice that reached significance at 0.63 mg/kg and the absence of effect in transgenic mice. *p < .05, two-tailed Wilcoxon MPSR test.

Hypothermia. RU24969 caused a dose-related drop in wild-type mouse body temperature that reached significance from 0.63 mg/kg on (Fig. 9A). A less pronounced drop was observed in transgenic mouse body temperature, which reached significance from 2.5 mg/kg on (Fig. 9B). Body temperature was not significantly influenced by the vehicle, as shown in Fig. 9. After a single injection of RU 24969 at 2.5 mg/kg, a significant drop in body temperature was observed in wild-type but not in transgenic mice (Fig. 10). The selective 5-HT_1A antagonist WAY100635 (2.5 mg/kg) but not the selective 5-HT_1B receptor antagonist GR127935 (2.5 mg/kg) significantly blocked the hypothermic effect of RU24969 induced in wild-type mice (p < .05 and p > .05, respectively, two-tailed Mann-Whitney U test) (Fig. 10). GR127935 and WAY100635 per se did not affect body temperature in comparison with vehicle (p > .05, two-tailed Mann-Whitney U test).

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Drop in esophageal temperature of wild-type (A) and transgenic (B) mice after s.c. injection of increasing doses of RU24969 (solid columns) or vehicle (open columns) (mean ± S.D.; n = 5). A, note that the drop in body temperature of wild-type mice occurs at lower dose than in transgenic mice (B). *p < .05, two-tailed Wilcoxon MPSR test.

View larger version (22K): [in this window] [in a new window]   Fig. 10.   Drop in esophageal temperature after administration of a 5-HT1B antagonist (GR127934, 2.5 mg/kg) or a 5-HT1A antagonist (WAY100635, 2.5 mg/kg) before the administration of RU24969 (2.5 mg/kg) (mean ± S.D.; n = 6). Note that WAY100635, but not GR127934, completely blocked the hypothermia induced by RU24969 in wild-type mice. *p < .05, two-tailed Mann-Whitney U test. Dotted columns, wild-type mice; gray columns, transgenic mice.

	Discussion

Top Summary Introduction Experimental Procedures Results Discussion References

An animal model to study the h5-HT_1B receptor in vivo was generated by a "knock-in" method; by humanizing the mouse locus, mice were generated that express the h5-HT_1B receptor instead of its murine counterpart from the same genetic locus. So far, functional studies of 5-HT_1B receptors of relevance to human application have been hampered by the differences in the pharmacology between human and rodent receptors.

The "humanized" mice described in the present study developed and lived apparently normally.

Localization and Densities of h5-HT_1B Receptor mRNA and Binding Sites Expressed in Transgenic Mouse Brain. The anatomic patterns and relative densities of h5-HT_1B and m5-HT_1B receptor mRNA throughout the brain of transgenic and wild-type mouse were completely similar; therefore, there is no indication that the spatial (and temporal) regulation of transcription from the 5-HT_1B gene is altered in transgenic animals (Table 1, Fig. 2, A and B). The anatomic patterns observed in this study also parallel a previous ISHH study in wild-type mouse brain (Boschert et al., 1994).

Pharmacological Characterization of h5-HT_1B Receptors Expressed in Transgenic Mouse Brain. The pIC₅₀ values derived from the radioligand binding inhibition study with various compounds in transgenic mouse and human substantia nigra brain sections were identical (Table 3). Inhibition of [³H]GR125743 binding by nine compounds was investigated. The pIC₅₀ values that were determined in this study using autoradiography on brain sections of transgenic mice, human substantia nigra, and wild-type mice are in agreement with binding affinities determined on membrane preparations of cloned human and rodent 5-HT_1B receptor expressed in cells, respectively (Hamblin et al., 1992; Bach et al., 1993; Leysen et al., 1996; Zgombick et al., 1997). The present pharmacological characterization demonstrates that after being expressed in the mouse brain, the human receptor shows the same pharmacological profile as the 5-HT_1B receptor in human brain, which is indeed different from the pharmacological profile of the 5-HT_1B receptor in mouse brain. The present study is also the first autoradiographic characterization of [³H]GR125743 binding sites in human tissue.

Functional Coupling of h5-HT_1B Receptors Expressed in Transgenic Mouse Brain to G Proteins. The observed stimulation of [³⁵S]GTPS binding by 5-CT, 5-HT, and alniditan in transgenic mice shows that the coupling between the h5-HT_1B receptor and mouse G protein is possible. The higher percentage of stimulation measured with 5-HT and 5-CT in wild-type animals is probably a consequence of the higher receptor densities observed in wild-type mice; however, it could also reflect a less efficient coupling between a human receptor and a mouse G protein. The higher percentages of stimulation observed in substantia nigra compared with globus pallidus are probably the consequence of a higher receptor density in the former brain area.

Absence of Modulation of Electrically Evoked [³H]5-HT Release by SB224289 in Slices of Transgenic Mice. The function of the 5-HT_1B receptor was measured in vitro by studying its effects on [³H]5-HT release from tissue slices of cerebral cortex, hippocampus, and striatum. The ability of the selective 5-HT_1B receptor antagonist SB224289 to potentiate [³H]5-HT release from electrically stimulated guinea pig cerebral cortical slices has recently been shown (Selkirk et al., 1998; M.H.M.B., I. Lenaerts and J.E.L., unpublished observations). Here, we demonstrate that SB224289 was also able to enhance electrically stimulated [³H]5-HT release from cerebral cortex and hippocampus in the wild-type mouse (Fig. 7). It did so in the absence of exogenously added agonists, indicating that endogenous 5-HT already exerts an inhibiting influence on the stimulated release. Such a tonic inhibition by endogenous 5-HT may explain the observation that agonists were not able to further inhibit the electrically induced [³H]5-HT release in the wild-type mice. In the transgenic mice, basal and electrically stimulated [³H]5-HT release could not be modulated; neither the 5-HT_1B agonist (alniditan) nor the 5-HT_1B antagonist (SB224289) had an effect on the release from the cerebral cortex or hippocampus slices, which is in contrast to the observations in brain slices of wild-type mice.

In Vivo Functional Responses to RU24969 in Transgenic Mice. The 5-HT_1B agonist RU24969 did not induce hyperlocomotion in the transgenic mice. It is noteworthy that RU24969 displays a 10 times lower affinity for h5-HT_1B receptor (Table 3; pIC₅₀ = 7.29) than for m5-HT_1B receptor (Table 3; pIC₅₀ = 8.15). We attempted to overcome this problem by increasing the doses of RU24969. Even at higher doses of RU24969 (20 and 40 mg/kg), no increase in locomotor activity was observed in transgenic animals, but at 40 mg/kg, the hyperlocomotion also was not present in wild-type mice, probably due to nonspecific sedative effects of the drug at a high dosage (unpublished observation). To the best of our knowledge, the mechanism of action by which RU24969 increases locomotor activity is far from clear (Tricklebank et al., 1986). Moreover, RU24969 is the only 5-HT_1B receptor agonist that was reported to increase locomotor activity; experiments with various other compounds failed.

Conclusions. The present in vitro and in vivo characterization studies demonstrate a complete replacement of the mouse receptor by its human receptor homolog and a functional coupling to G proteins; however, we could not reliably demonstrate downstream functional effects.