The Journal of Neuroscience, March 15, 2001, 21(6):2178–2185 Functional Consequences of 5-HT Transporter Gene Disruption on

Receptor-Mediated Regulation of Dorsal Raphe and
Hippocampal Cell Activity
Clotilde Mannoury la Cour,1 Claudette Boni,1 Naı¨ma Hanoun,1 Klaus-Peter Lesch,2 Michel Hamon,1 and
Laurence Lanfumey
1Institut National de la Sante´ et de la Recherche Me´dicale U288, Neuropsychopharmacologie Mole´culaire, Cellulaire etFonctionnelle, Faculte´ de Me´decine Pitie´-Salpeˆtrie`re, 75634 Paris Cedex 13, France, and 2Department of Psychiatry,University of Wu¨rzburg, 97080 Wu¨rzburg, Germany The consequences of the absence of 5-HT reuptake on the trasted with those obtained with hippocampal slices in which 5-carboxamidotryptamine was equipotent to hyperpolarize dorsal raphe nucleus and the hippocampus of knock-out mice CA1 pyramidal neurons in both mutant and wild-type mice. As lacking the serotonin transporter (5-HTT). Extracellular record- expected from their mediation through 5-HT ings showed that application of selective 5-HT reuptake inhib- effects of ipsapirone and 5-carboxamidotryptamine were com- itors such as paroxetine and citalopram onto brainstem slices petitively inhibited by the selective 5-HT resulted in a concentration-dependent inhibition of 5-HT neu- 100635 in both groups. These data showed that 5-HTT gene ron firing in the dorsal raphe nucleus of wild-type 5-HTTϩ/ϩ knock-out induced a marked desensitization of 5-HT mice, but not 5-HTTϪ/Ϫ mutants. By contrast, the 5-HT ceptors in the dorsal raphe nucleus without altering postsyn- receptor agonists ipsapirone and 5-carboxamidotryptamine in- receptor functioning in the hippocampus. Similar- hibited the discharge in both groups. However, the potency ities between these changes and those evoked by chronic of these agonists was markedly decreased (by ϳ55- and ϳ6- treatment with 5-HT reuptake inhibitors emphasize the exis- fold, respectively) in 5-HTTϪ/Ϫ compared with 5-HTTϩ/ϩ an- imals. Similarly, intracellular recordings showed that the po- tency of 5-carboxamidotryptamine to hyperpolarize 5-HT Key words: 5-HT transporter knock-out mice; 5-HT neurons in the dorsal raphe nucleus was significantly lower tors; dorsal raphe nucleus; hippocampus; desensitization; in in 5-HTTϪ/Ϫ than in 5-HTTϩ/ϩ animals. These data con- The involvement of the serotoninergic system in major psychiatric 1996) allowed the generation of an animal model with targeted diseases, in particular mood disorders such as depression, is a well disruption of this gene by homologous recombination (Bengel et established clinical feature (Asberg et al., 1976; Cryan and Leo- al., 1998). Indeed, the deletion of exon 2 results in an inactive nard, 2000). Accordingly, to date, the most frequently used anti- gene and the complete absence of 5-HT reuptake activity in the depressants are the selective serotonin reuptake inhibitors homozygous 5-HTTϪ/Ϫ mice. No apparent developmental alter- (SSRIs), which act on the Naϩ/ClϪ-dependent 5-HT transporter ations were noted in the null mutant mice, suggesting that major (5-HTT) (Graham et al., 1989; Lesch, 1997). In the CNS, 5-HTT compensatory mechanisms occur in these animals during embry- seems to be essentially localized on serotoninergic neurons, at the onic and subsequent neurodevelopment (Bengel et al., 1998).
level of somas, dendrites, axons, and terminals (Hensler et al., However, marked depletions of 5-HT and of its metabolite 1994; Sur et al., 1996; Tao-Cheng and Zhou, 1999), and only a 5-hydroxyindoleacetic acid in brain evidenced that adaptive minor glial expression of this protein has also been reported by changes in 5-HT neurotransmission do occur in 5-HTTϪ/Ϫ mu- some authors (Hirst et al., 1998; Pickel and Chan, 1999). In any tants (Bengel et al., 1998; Li et al., 1999; Fabre et al., 2000).
case, it is well established that the 5-HTT is responsible for the Previous studies using biochemical and neuroendocrinological primary mechanism of 5-HT inactivation in the CNS (Lesch, approaches further investigated adaptive changes in 5-HT neu- rotransmission in 5-HTTϪ/Ϫ mutants with particular attention to Elucidation of the murine 5-HTT gene sequence (Chang et al., 5-HT receptors. Indirect evidence of desensitization and down- regulation of 5-HT1A autoreceptors in the dorsal raphe nucleus Received Aug. 7, 2000; revised Dec. 22, 2000; accepted Jan. 4, 2001.
(DRN) (Li et al., 1999; Fabre et al., 2000) and 5-HT2A receptors This research was supported by the Institut National de la Sante´ et de la in the striatum and cerebral cortex (Rioux et al., 1999) has thus Recherche Me´dicale and Bristol-Myers Squibb Foundation (Unrestricted Biomed- ical Research Grant Program). C.M.C. was a recipient of a Fondation pour la been reported in knock-out mice. Interestingly, similar changes in Recherche Me´dicale fellowship during performance of this work. We are grateful to these receptors have previously been shown to occur after chronic pharmaceutical companies (Lundbeck, Pierre Fabre, SmithKline Beecham, Troponwerke-Bayer, and Wyeth-Ayerst) for generous gifts of drugs.
blockade of 5-HT reuptake by SSRI (Chaput et al., 1986; Sanders- Correspondence should be addressed to C. Mannoury la Cour, Institut National de Bush et al., 1989; Jolas et al., 1994; Kreiss and Lucki, 1995; Le la Sante´ et de la Recherche Me´dicale U 288, Neuropsychopharmacologie Mole´culaire, Poul et al., 1995, 2000), thereby suggesting that the 5-HTTϪ/Ϫ Cellulaire et Fonctionnelle, Faculte´ de Me´decine Pitie´-Salpeˆtrie`re 91, Boulevard de l’Hoˆpital, 75634 Paris Cedex 13, France. E-mail:
mutant mouse can be considered as a model of whole-life treat- Copyright 2001 Society for Neuroscience 0270-6474/01/212178-08$15.00/0 ment with these drugs. Interestingly, Le Poul et al. (2000) recently Receptors in 5-HT Transporter Knock-Out Mice J. Neurosci., March 15, 2001, 21(6):2178–2185 2179
DRN and pyramidal cells in the CA1 area of the hippocampus were recorded in current-clamp mode with 3 M KCl-filled electrodes (50 – 80 M⍀), while brain slices were superfused with ACSF (Corradetti et al., 1998). Electrical signals were amplified with an Axoclamp 2A (Axon These data led us to investigate further the functional status of Instruments, Foster City, CA) and displayed on an oscilloscope and a 5-HT1A receptors in these two areas in 5-HTTϪ/Ϫ mutants chart recorder. Traces were stored in a digital tape recorder (DTR 1202; versus wild-type mice. For this purpose, both extracellular and BioLogic; 48 kHz sampling frequency) and a computer using pClamp6 intracellular electrophysiological recordings of 5-HT software (3–10 kHz sampling frequency; Axon Instruments) for off-line measurements. Only neurons with stable resting membrane potential ing neurons in brain slices were used to quantitatively assess their (range, Ϫ50 to Ϫ90 mV) and input resistance (R CA1 neurons and 100 –500 M⍀ for DRN cells) throughout the recording session were included in the analysis. Membrane potential in response to hyperpolarizing and depolarizing current pulses of 50/100 pA increments MATERIALS AND METHODS
(range, Ϫ900 to ϩ500 pA) was measured before, during, and after tissue superfusion with drugs added to the ACSF. To draw concentration– Experiments were performed using homozygous 5-HTTϪ/Ϫ, heterozy- response curves for the 5-HT1 agonist 5-carboxamidotryptamine (5-CT), gous 5-HTTϩ/Ϫ, and wild-type 5-HTTϩ/ϩ littermates born from het- the membrane potential was recorded while slices were superfused erozygous mutants of C57BL6 genetic background. Genotyping was with increasing concentrations of this ligand. Preliminary experiments performed as described by Bengel et al. (1998). Animals were used at 2 (data not shown) demonstrated that for a given cell, consecutive appli- months of age when their body weight in each genotype equally ranged cations of increasing concentrations of 5-CT produced cumulative between 20 and 25 gm. After weaning and sexing, males and females concentration-dependent responses with a maximal effect equal to that were housed separately in groups of six to eight animals per cage and obtained with application of a single saturating concentration. Nonlinear maintained under standard laboratory conditions (22 Ϯ 1°C; 60% rela- regression fitting was performed using Prism 2.0 (GraphPad) software tive humidity; 12 hr light/dark cycle; food and water available ad libitum).
facilities for the determination of concentration-dependent hyperpolar- In addition, some experiments were performed using CD1, C57BL6, and ization and decrease in Rin caused by 5-CT.
c129 control mice provided by the Centre d’Elevage R. Janvier (Le Genest-St. Isle, France) and IFFA Credo (Lyon, France), respectively.
Procedures involving animals and their care were conducted in con- All data are given as means Ϯ SEM. Extracellular and intracellular formity with the institutional guidelines that are in compliance with recording data were analyzed by one-way ANOVA and, in case of national and international laws and policies (council directive number significance ( p Ͻ 0.05), the F test for significant treatment effects was 87– 848, 19 October 1987, Ministe`re de l’Agriculture et de la Foreˆt, followed by the two-tailed Student’s t test to compare the experimental Service Ve´te´rinaire de la Sante´ et de la Protection Animale, permissions groups with their controls. A value of p Ͻ 0.05 was considered to be Preparation of slices of DRN and dorsal hippocampus. Mice were decap- The following drugs were used: ipsapirone (Bayer-Troponwerke, Cologne, itated, and the brains were rapidly removed and immersed in an ice-cold Germany), 5-CT (Research Biochemicals, Natick, MA), paroxetine (Smith- Krebs’ solution, bubbled continuously with an O Kline Beecham, Harlow, UK), citalopram (Lundbeck, Copenhagen, (95:5%). A block of tissue containing the DRN or the dorsal hippocam- N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N- pus was cut into sections (350- to 400-␮m-thick) in the same ice-cold (2-pyridinyl)cyclohexane carboxamide (WAY 100635; Wyeth-Ayerst, Krebs’ solution using a vibratome (Corradetti et al., 1998). Brainstem or hippocampus slices were then immediately incubated at room tempera- ture (20 –23°C) for at least 1 hr in an artificial CSF (ACSF) of the following composition (mM): NaCl 126, KCl 3.5, NaH2PO4 1.2, MgCl2 In both the DRN and the hippocampus, electrophysiological re- 2 2, NaHC O3 25, D-glucose 11, maintained at pH 7.3 by continuous bubbling with O2–CO2 mixture. A slice of either the DRN or cordings under the various pharmacological conditions tested did the CA1 hippocampal area was then placed on a nylon mesh, completely not reveal any significant differences between males and females of submerged in a small chamber, and superfused continuously with oxy- the homozygous 5-HTTϪ/Ϫ, heterozygous 5-HTTϩ/Ϫ, or wild- genated ACSF (34°C) at a constant flow rate of 2–3 ml/min (Corradetti type phenotype. Accordingly, both males and females were used Extracellular recordings of serotoninergic neurons in the dorsal raphe indifferently in the experiments reported herein.
nucleus. Extracellular recordings were made with glass microelectrodes filled with 2 M NaCl (10 –15 M⍀). Cells were identified as 5-HT neurons Extracellular recordings of DRN 5-HT neurons
according to the following criteria: biphasic action potentials and slow and regular pattern of discharge (1.5–2.5 spikes/sec) (Trulson and Fred- Because generation of the 5-HTTϪ/Ϫ knock-out model required erickson, 1987; Jacobs and Azmitia, 1992). Firing was evoked in the otherwise silent neurons by adding the ␣ the use of three different strains of mice (c129, CD1, and C57BL6) ephrine (3 ␮M) to the superfusing ACSF (VanderMaelen and Aghaja- (Bengel et al., 1998), some heterogeneity in the genetic background nian, 1983). Baseline activity was recorded for 5–10 min before the might have still existed, thereby accounting for possible variations application of drugs via a three-way tap system that allowed complete in the electrophysiological characteristics of DRN 5-HT neurons in exchange of fluids within 2 min of arrival of a new solution. The electrical signals were fed into a high-input impedance amplifier (VF180; Bio- 5-HTϪ/Ϫ mutants compared with wild-type animals of these Logic, Claise, France), an oscilloscope, and an electronic ratemeter strains. To directly assess this possibility, the spontaneous dis- triggered by individual action potentials connected to an analog-to- charge frequency of DRN 5-HT neurons was compared in paired digital converter and a personal computer (Haj-Dahmane et al., 1991).
control 5-HTTϩ/ϩ mice and in c129, CD1, and C57BL6 mice.
The integrated firing rate was computed and recorded graphically as Indeed, the baseline firing rate of 5-HT neurons was similar in the consecutive 10 sec samples. The effect of a given drug was evaluated by comparing the mean discharge frequency during the 2 min before its four groups: c129, 1.72 Ϯ 0.19 spikes/sec (mean Ϯ SEM, n ϭ 8); addition to the superfusing ACSF with that recorded at the peak of the CD1, 1.87 Ϯ 0.17 spikes/sec (n ϭ 7); C57BL6, 1.49 Ϯ 0.11 spikes/ action of the drug, i.e., 3–10 min after starting the drug infusion. When sec (n ϭ 12), and 5-HTTϩ/ϩ, 1.89 Ϯ 0.15 spikes/sec (n ϭ 12).
an agonist was applied in the presence of an antagonist, the effect of the Furthermore, the baseline firing rate of DRN 5-HT neurons was agonist was compared with the baseline firing rate and with the discharge frequency recorded during superfusion with the antagonist alone.
also not significantly different from these values in heterozygous Intracellular recordings of serotoninergic neurons in the dorsal raphe 5-HTTϩ/Ϫ, 1.88 Ϯ 0.19 spikes/sec (n ϭ 6) and homozygous nucleus and pyramidal neurons in the hippocampus. 5-HT neurons in the 5-HTTϪ/Ϫ mutants, 1.66 Ϯ 0.18 spikes/sec (n ϭ 10).
2180 J. Neurosci., March 15, 2001, 21(6):2178–2185
Receptors in 5-HT Transporter Knock-Out Mice Figure 1. Effects of paroxetine and and wild-type mice. A, Integrated firing centrations of paroxetine (top) or cita- lopram (bottom) on the electrical activ- mice. B, Concentration-dependent inhi- bition by paroxetine (top) or citalopram (bottom) of the firing of DRN 5-HT neu- induced inhibition is expressed as a per- centage of the baseline firing rate. Each cells. The dotted lines illustrate the deter- Effects of the SSRIs paroxetine and citalopram Ipsapirone also inhibited the discharge of DRN 5-HT cells in Like that previously reported under similar conditions in rats (Le the 5-HTTϩ/Ϫ and 5-HTTϪ/Ϫ mutants, but within higher con- Poul et al., 1995), addition of increasing concentrations of parox- centration ranges than in wild-type animals. Although the in- etine into the ACSF superfusing brainstem slices resulted in a crease in the EC50 value of ipsapirone in heterozygous 1A receptor-mediated concentration-dependent inhibition 50 ϭ 115.3 Ϯ 7.3 nM; n ϭ 9) was not of the firing of DRN 5-HT neurons in wild-type 5-HTTϩ/ϩ mice significant, that in homozygous 5-HTTϪ/Ϫ mutants (EC50 ϭ (Fig. 1). A similar effect was noted in heterozygous 5-HTTϩ/Ϫ 3.5 Ϯ 1.1 ␮M; n ϭ 10) was highly significant ( p Ͻ 0.001), indicating an ϳ55-fold decrease in the potency of the 5-HT 2.10 Ϯ 0.43 ␮M (mean Ϯ SEM, n ϭ 7), did not significantly differ receptor agonist in the latter group compared with wild-type from that in wild-type controls, 3.70 Ϯ 0.73 ␮M (n ϭ 7) (Fig. 1).
controls. In spite of these differences, complete blockade of the By contrast, the same treatment applied to brainstem slices from discharge of DRN 5-HT cells could be achieved in the three 5-HTTϪ/Ϫ mice produced only a minor (less than or equal to groups, but with different concentrations of ipsapirone (1 ␮M in Ϫ15%), concentration-independent, reduction in the firing rate 5-HTTϩ/ϩ and 5-HTTϩ/Ϫ mice, 100 ␮M in 5-HTTϪ/Ϫ mice) of DRN 5-HT cells, even at paroxetine concentration as high as (Fig. 2). Similar results were found with 5-CT as 5-HT1 agonist.
Thus, 5-CT (1 nM to 10 ␮M) induced a concentration-dependent Similar findings were obtained with citalopram, which potently decrease in the firing rate of DRN 5-HT neurons with a signifi- inhibited, in a concentration-dependent manner, the discharge of cantly ( p Ͻ 0.001) lower potency in knock-out (EC50 ϭ 52.7 Ϯ 3.6 nM; n ϭ 10) than in wild-type mice (EC n ϭ 5), but remained essentially inactive in homozygous 10). However, the relative decrease in 5-CT potency in the 5-HTTϪ/Ϫ mutants (less than or equal to Ϫ10% in the firing rate mutants (by approximately sixfold) was less than that noted for at 0.1–30 ␮M citalopram) (Fig. 1).
ipsapirone, as illustrated by the shift to the right of concentra- tion–response curves, which was of much larger amplitude with Effect of 5-HT1A autoreceptor stimulation the latter compared with the former agonist (Fig. 2). In any case, As expected from the stimulation of somatodendritic 5-HT1A as expected from their mediation through 5-HT1A autoreceptors, autoreceptors (Haj-Dahmane et al., 1991), the addition of the the inhibitory effects of both ipsapirone and 5-CT were prevented 5-HT1A receptor agonist ipsapirone into the ACSF superfusing by the selective 5-HT1A antagonist WAY 100635 (1–3 nM) in both brainstem slices resulted in a concentration-dependent inhibition of the firing of DRN 5-HT neurons in wild-type 5-HTTϩ/ϩ mice (Fig. 2). Similar effects were noted in c129, CD1, and C57BL6 Effects of 5-HT1A receptor blockade by WAY 100635 mice, and the EC50 value of ipsapirone was not significantly Further characterization of ipsapirone–WAY 100635 interactions different in these four murine strains: c129, 61.3 Ϯ 6.1 nM consisted of investigating the concentration-dependent inhibition (mean Ϯ SEM, n ϭ 9); CD1, 54.1 Ϯ 3.5 nM (n ϭ 9); C57BL6, of DRN 5-HT neuron firing by ipsapirone in the absence or the 44.9 Ϯ 6.1 nM (n ϭ 9); and 5-HTTϩ/ϩ, 63.1 Ϯ 7.4 nM (n ϭ 10).
presence of a fixed concentration (2 nM) of the 5-HT1A receptor Receptors in 5-HT Transporter Knock-Out Mice J. Neurosci., March 15, 2001, 21(6):2178–2185 2181
Figure 2. Concentration-dependent inhibition by ipsapirone or 5-CT of the electrical activity of DRN 5-HT neurons in 5-HTT knock-out and wild-type mice. Prevention by WAY 100635. A, Integrated firing rate histograms (in spikes per 10 sec) showing the inhibitory effect of ipsapirone and its prevention by WAY 100635, on the electrical activity of DRN 5-HT cells in 5-HTTϪ/Ϫ and 5-HTTϩ/Ϫ mutants compared with 5-HTTϩ/ϩ wild-type mice (top). The effect of 5-CT (bottom), and its prevention by WAY 100635, are illustrated in 5-HTTϩ/ϩ and 5-HTTϪ/Ϫ mice.
Histograms are from different neurons. B, Concentration-dependent inhibition by ipsapirone (top) or 5-CT (bottom) of the firing of DRN 5-HT neurons in 5-HTTϩ/ϩ, 5-HTTϪ/Ϫ, and/or 5-HTTϩ/Ϫ mice. Agonist-induced inhibition is expressed as a percentage of the baseline firing rate.
Each point is the mean Ϯ SEM of data obtained from three to seven individual cells. The dotted lines illustrate the increase in the EC50 values (abscissa) of ipsapirone and 5-CT in 5-HTTϪ/Ϫ compared with 5-HTTϩ/ϩ mice. *p Ͻ 0.05; **p Ͻ 0.01; ***p Ͻ 0.001 as compared with the corresponding inhibition in 5-HTTϩ/ϩ and 5-HTTϩ/Ϫ mice.
antagonist. Data in Figure 3 show that WAY 100635 produced a Intracellular recordings of DRN 5-HT neurons and
shift to the right of the ipsapirone curve in wild-type as well as hippocampal pyramidal neurons
mutant mice, as expected from competitive inhibition of the effect of ipsapirone by WAY 100635. Calculation of the IC50 value of In the absence of drugs, 5-HT cells recorded in 5-HTTϩ/ϩ (n ϭ WAY 100635 from these curves yielded 0.065 Ϯ 0.015, 0.078 Ϯ 6) as well as in null mutants (n ϭ 5), exhibited similar membrane 0.021, and 0.140 Ϯ 0.035 nM (means Ϯ SEM; n Ն 5 for each value) potential and Rin ranging from Ϫ66 to Ϫ83 mV and 158 to 255 in 5-HTTϩ/ϩ, 5-HTTϩ/Ϫ, and 5-HTTϪ/Ϫmice, respectively.
M⍀, respectively. Bath-applied ipsapirone evoked both a In a second series of experiments, the concentration-dependent concentration-dependent membrane hyperpolarization (with a prevention by WAY 100635 (5 pM-5 nM) of the inhibitory effect of maximal response of Ϫ4.50 Ϯ 0.76 mV, n ϭ 4, in the presence of a fixed concentration of ipsapirone (300 nM) on the discharge of 300 nM ipsapirone) and a decrease in Rin (down to 62% of DRN 5-HT neurons was compared in 5-HTTϩ/ϩ and baseline value) of DRN 5-HT neurons in wild-type mice (Fig.
5-HTTϪ/Ϫ mice. Calculations of the IC50 of the 5-HT1A antag- 4A). By contrast, neither the membrane potential nor the Rin of onist yielded values in the same range as those calculated from DRN 5-HT neurons in slices from 5-HTTϪ/Ϫ mutants were the previous series of experiments and did not significantly differ affected by ipsapirone, even at a concentration as high as 30 ␮M between wild-type (0.093 Ϯ 0.032 nM; n ϭ 5) and knock-out (Fig. 4A). Different results were obtained with the other agonist (0.045 Ϯ 0.014 nM; n ϭ 4) animals.
tested, 5-CT, because this compound, in contrast to ipsapirone, 2182 J. Neurosci., March 15, 2001, 21(6):2178–2185
Receptors in 5-HT Transporter Knock-Out Mice Figure 3. Competitive inhibition by WAY 100635 of the negative effect of ipsapirone on the firing of DRN 5-HT neurons in 5-HTTϪ/Ϫ and 5-HTTϩ/Ϫ mutants compared with 5-HTTϩ/ϩ wild-type mice. Experiments were as described in the legend to Figure 2 except that the effects of various concentrations of ipsapirone were tested in the absence (black symbols) or the presence (open symbols) of 2 nM WAY 100635. Inhibition caused by ipsapirone is expressed as a percentage of baseline firing rate. Each point is the mean Ϯ SEM of data obtained from five to seven individual cells. *p Ͻ 0.05; **p Ͻ 0.01; ***p Ͻ 0.001 as compared with corresponding data in the absence of WAY 100635.
produced a concentration-dependent membrane hyperpolariza- well as wild-type (data not shown) mice. Concentration- tion of DRN 5-HT neurons in both knock-out and wild-type mice dependent prevention by WAY 100635 (0.3–10 nM) of the mem- (Fig. 4B,C). As expected from the higher agonist efficacy of 5-CT brane hyperpolarization induced by 300 nM 5-CT showed no compared with ipsapirone (Bockaert et al., 1987; Hoyer et al., differences between wild-type (IC50 ϭ 0.96 Ϯ 0.04 nM; n ϭ 3) and 1994), maximal membrane hyperpolarization induced by the knock-out (IC50 ϭ 1.07 Ϯ 0.06 nM; n ϭ 3) mice (Fig. 5C).
former agonist was (ϳ2.5-fold) larger than that observed with the latter in wild-type animals (Fig. 4C). Concentration-dependent DISCUSSION
curves showed no difference in 5-CT-induced maximal mem- The present study showed that the lack of 5-HT reuptake because brane hyperpolarization in 5-HTTϪ/Ϫ versus 5-HTTϩ/ϩ mice; of the deletion of exon 2 in the 5-HTT gene (Bengel et al., 1998) however, the potency of this agonist was significantly ( p Ͻ 0.001) induces major alterations in central 5-HT neurotransmission. In 50 ϭ 89.6 Ϯ 2.9 nM; n ϭ 3) than in erts a key role in the modulation of 5-HT tone (Hamon, 1997), is 50 ϭ 11.9 Ϯ 1.0 nM; n ϭ 3) animals (Fig. 4C) In both groups, the effects of 5-CT (300 nM) were completely prevented deeply desensitized in the knock-out 5-HTTϪ/Ϫ mice. However, by WAY 100635 (10 nM), which, on its own, affected neither the such a functional adaptation does not extend to all 5-HT1A receptors in brain because those located postsynaptically in the in of DRN 5-HT cells (Fig. 4 B; data hippocampus were found to exhibit the same characteristics in Like that previously reported in rats (Rigdon and Wang, 1991; Like that noted for DRN 5-HT cells, no significant differences Le Poul et al., 1995), the increase in extracellular 5-HT concen- were found in the membrane potential (range, Ϫ53 to Ϫ78 mV) trations within the DRN of brainstem slices exposed to SSRI and the Rin value (range, 48–133 M⍀) of CA1 pyramidal cells (paroxetine, citalopram) was found to trigger a 5-HT1A between 5-HTTϪ/Ϫ mutant and 5-HTTϩ/ϩ wild-type mice. The autoreceptor-mediated inhibition of DRN 5-HT cell firing in addition of increasing concentrations (30 nM to 1 ␮M) of 5-CT wild-type mice. This response offered a relevant model to further into the ACSF superfusing hippocampal slices from wild-type assess the lack of 5-HTT in the knock-out animals, and indeed, as mice elicited a hyperpolarization of cell membrane (maximal expected, neither paroxetine nor citalopram were able to produce response, Ϫ6.55 Ϯ 0.41 mV with 300 nM 5-CT; n ϭ 8) and a a concentration-dependent inhibition of DRN 5-HT cell firing in decrease in Rin value (Ϫ44.2% with 300 nM 5-CT) (Fig. 5A,B).
5-HTTϪ/Ϫ mutants. These electrophysiological data further con- These effects were reversible with recovery of predrug values firmed previous autoradiographic and biochemical results show- within ϳ15 min after removal of 5-CT from the superfusing ing the complete absence of the SSRI molecular target, i.e., the ACSF (Fig. 5A). As illustrated in Figure 5B, 5-CT-induced effects 5-HTT, in the homozygous mutants (Bengel et al., 1998; Fabre et were clearly concentration-dependent, with an EC50 value of al., 2000). In contrast, experiments performed with tissues from 41.0 Ϯ 4.0 nM (n ϭ 6). Similar effects were noted on CA1 heterozygous 5-HTTϩ/Ϫ mice showed that paroxetine inhibited pyramidal cells in hippocampal slices from 5-HTTϪ/Ϫ animals, DRN 5-HT cell firing with the same potency in these mutants as and indeed the maximal hyperpolarization (Ϫ6.05 Ϯ 0.50 mV; in wild-type animals, although the density of 5-HT transporter n ϭ 9) and decrease in Rin (Ϫ43.7%) in response to 300 nM 5-CT binding sites was only half in the former compared with the latter (Fig. 5), and the potency of this drug (EC50 ϭ 51.1 Ϯ 5.0 nM; n ϭ group (Fabre et al., 2000). Interestingly, Bengel et al. (1998) 9) to trigger these effects, were not significantly different in the reported that in vitro synaptosomal [3H]5-HT uptake was also homozygous mutants compared with wild-type mice.
unchanged in 5-HTTϩ/Ϫ compared with wild-type animals. It As expected from effects mediated through 5-HT1A receptor can thus be hypothesized that adaptive changes in 5-HTT intrin- stimulation, both the membrane hyperpolarization and the de- sic activity very probably occur to compensate for the (partial) crease in Rin value caused by 300 nM 5-CT could be completely loss of 5-HTT protein in heterozygous 5-HTTϩ/Ϫ mutants.
prevented by bath application of 10 nM of the selective 5-HT1A One of the most interesting observations made in our studies is receptor antagonist WAY 100635 (Fig. 5A). On its own, WAY that spontaneous 5-HT neuron firing in brainstem slices was not 100635 affected neither the membrane potential nor the Rin value altered in 5-HTTϪ/Ϫ mutants compared with wild-type mice.
of CA1 pyramidal neurons in homozygous mutant (Fig. 5A) as Because the electrophysiological activity of DRN 5-HT cells is Receptors in 5-HT Transporter Knock-Out Mice J. Neurosci., March 15, 2001, 21(6):2178–2185 2183
Figure 4. Differential effects of ipsapirone and 5-CT on intracellularly recorded DRN 5-HT neurons in 5-HTT knock-out and wild-type mice. A, Chart recordings of membrane potential of a DRN 5-HT neuron in a brainstem slice from a 5-HTTϩ/ϩ (top) versus a 5-HTTϪ/Ϫ (bottom) mouse. Each successive concentration of ipsapirone was applied for 4 min. B, Same as in A, except that 5-CT (300 nM for 4 min) was substituted for ipsapirone. Bottom recording shows the prevention by 10 nM WAY 100635 of 5-CT-induced hyperpolarization of the same cell as that corresponding to the middle recording. Downward and upward rapid deflections in A and B are electrotonic cell membrane responses to constant current steps (Ϫ200 to ϩ200 pA) injected through the recording electrode. Similar data were obtained in at least five cells in each group. C, Concentration–response curves of 5-CT-induced hyperpolarization of DRN 5-HT neurons in 5-HTTϩ/ϩ and 5-HTTϪ/Ϫ mice. Each point is the mean Ϯ SEM of data obtained in three to five cells for each concentration of 5-CT. Dotted lines point to the EC50 values (abscissa). **p Ͻ 0.01; *p Ͻ 0.05 compared with respective hyperpolarization in the 5-HTTϩ/ϩ group.
negatively controlled by extracellular 5-HT acting at 5-HT1A 5-HT neuron firing in brainstem slices from mice of various autoreceptors (Sprouse and Aghajanian, 1987; Haj-Dahmane et strains, including 5-HTTϩ/ϩ animals. With respect to the inhib- al., 1991), one would have expected that the lack of 5-HT re- itory effect of ipsapirone, heterozygous 5-HTTϩ/Ϫ mice did not uptake produces some reduction in their firing rate because of the significantly differ from wild-type mice (further supporting the resulting increase in extracellular 5-HT levels in 5-HTTϪ/Ϫ idea that compensatory changes occurred in these mutants, see mice. Indeed, using in vivo microdialysis, marked increases (by at above), whereas homozygous 5-HTTϪ/Ϫ mutants were much less least sixfold) in extracellular 5-HT levels were found in the sensitive to the drug. Indeed ipsapirone potency was ϳ55-fold substantia nigra (Fabre et al., 2000) and the striatum (Andrews et lower in the latter animals than in wild-type mice. Similar results al., 1998) of 5-HTTϪ/Ϫ compared with 5-HTTϩ/ϩ mice. No were found using 5-CT, except that the potency of this agonist to data have yet been published concerning extracellular 5-HT con- inhibit DRN 5-HT neuron firing was decreased by only approxi- centrations within the DRN, but it can be reasonably assumed mately sixfold in 5-HTTϪ/Ϫ compared with 5-HTTϩ/ϩ animals.
that they are also markedly enhanced in 5-HTTϪ/Ϫ mice, espe- This difference between the two agonists was as expected from cially because the DRN contains a high density of 5-HT reuptake their respective efficacy at 5-HT1A receptors, because it is well sites (Bengel et al., 1997; Rattray et al., 1999).
established (Kenakin, 1993) that reductions in receptor number Because the most probable explanation for the maintenance of and/or coupling, such as those affecting 5-HT1A autoreceptors in normal basal firing rate of DRN 5-HT cells in 5-HTTϪ/Ϫ mice is 5-HTTϪ/Ϫ mice (Fabre et al., 2000), decrease to a greater extent that 5-HT1A autoreceptor-mediated inhibitory control is altered, the response to a low-efficacy (partial) agonist such as ipsapirone we directly investigated the functional properties of DRN (Bockaert et al., 1987), than a high-efficacy (full) agonist such as 5-HT1A autoreceptors in these mutants. Like that previously observed in rats (Haj-Dahmane et al., 1991), bath application of Interestingly, in addition to that of 5-HT1A receptor agonists, ipsapirone induced a concentration-dependent inhibition of DRN the potency of baclofen, a GABA-B receptor agonist, to inhibit 2184 J. Neurosci., March 15, 2001, 21(6):2178–2185
Receptors in 5-HT Transporter Knock-Out Mice Figure 5. 5-CT-induced hyperpolariza- type mice. Prevention by WAY 100635.
A, Chart recordings of membrane poten- min after cessation of 5-CT application.
cording electrode. B, Concentration–re- concentration of 5-CT. Dotted lines point to the EC50 values (abscissa). C, Con- centration–response curves of the antag- Ϫ5.8 to Ϫ6.7 mV; response range for n ϭ 9 individual cells in each group). Each point is the mean Ϯ SEM of data obtained in three or four cells for each concentration of WAY 100635. The dotted lines indicate the IC50 values of WAY 100635 (abscissa) against 5-CT-evoked hyperpolarization.
the discharge of DRN 5-HT neurons, was also found to be rats (Le Poul et al., 1995, 2000). It has to be stressed, however, decreased in 5-HTTϪ/Ϫ versus 5-HTTϩ/ϩ mice (Mannoury La that 5-HTT is usually inhibited for only 2–3 weeks in pharmaco- Cour et al., 2000). Because both 5-HT1A and GABA-B receptors logical models, whereas it is completely inactivated for the whole share the same pool of G-proteins (Andrade et al., 1986), it can be life in knock-out animals, including at critical periods during inferred that possible alterations in this pool underlie their con- development when 5-HT can play specific actions on brain mat- uration (Emerit et al., 1992; Lotto et al., 1999).
Further analyses of the 5-HT1A-mediated responses by intra- Another key feature of chronic SSRI treatments is the differen- cellular recording of DRN 5-HT cells could not be performed tial fate of hippocampal postsynaptic 5-HT1A receptors versus with ipsapirone because this partial 5-HT1A agonist lost its ca- DRN 5-HT1A autoreceptors in rats subjected to such treatments.
pacity to hyperpolarize the cell membrane in 5-HTTϪ/Ϫ mu- Thus, in contrast to the latter receptors, those in the hippocampus tants. This led us to use the full 5-HT1 agonist, 5-CT, whose do not desensitize after chronic SSRI administration (Haddjeri et effects on the membrane potential and Rin also appeared to be al., 1998; Le Poul et al., 2000). To assess further the possible completely prevented by WAY 100635, as expected from their relevance of 5-HTT gene knock-out as a model of chronic 5-HTT mediation through 5-HT1A receptors (Hamon, 1997). Indeed, blockade by SSRI, we investigated the functional characteristics of 5-CT was still able to hyperpolarize the plasma membrane of 5-HT1A receptors on pyramidal cells in the CA1 area of the DRN 5-HT neurons in 5-HTTϪ/Ϫ mice, but with a significantly hippocampus in 5-HTTϪ/Ϫ mutants compared with wild-type lower potency than in wild-type animals. The differences between mice. 5-CT, rather than ipsapirone, was used in these experiments ipsapirone and 5-CT revealed by these intracellular recording because the partial agonist properties of the latter ligand produced experiments were also as expected from respective changes in the only minor, not reliably measurable, hyperpolarization of CA1 response to a partial and a full agonist (Bockaert et al., 1987; pyramidal neurons (L. Lanfumey, unpublished observations). Like Hoyer et al., 1994) after alterations in their shared receptors that observed in rats (Corradetti et al., 1998), 5-CT application (Kenakin, 1993) such as those observed in DRN 5-HT1A autore- onto mouse hippocampal slices produced both a hyperpolarization of the plasma membrane and a decreased Rin of CA1 pyramidal Previous studies in rats have shown that chronic impairment of cells that could be completely prevented by WAY 100635, demon- 5-HT reuptake by long-term SSRI treatment also induces a sig- strating their mediation through 5-HT1A receptors. Comparison of nificant desensitization of DRN 5-HT1A autoreceptors (Chaput the potency of 5-CT to induce these effects in 5-HTTϪ/Ϫ versus et al., 1986; Jolas et al., 1994; Kreiss and Lucki, 1995; Le Poul et 5-HTTϩ/ϩ mice revealed no difference between the two groups, al., 1995, 2000), thereby suggesting that similar mechanisms are indicating that postsynaptic 5-HT1A receptors were not desensi- responsible for this adaptive phenomenon in both SSRI-treated tized in the knock-out animals. In line with these observations, animals and 5-HTT knock-out mice. However, differences also Fabre et al. (2000) recently reported that 5-HT1A receptor-evoked exist between these two experimental models because in contrast [35S]GTP-␥-S specific binding was significantly decreased in the to that found in 5-HTTϪ/Ϫ mice (Fabre et al., 2000), DRN DRN but not the hippocampus in 5-HTTϪ/Ϫ compared with 5-HT1A autoreceptors are not downregulated in SSRI-treated Receptors in 5-HT Transporter Knock-Out Mice J. Neurosci., March 15, 2001, 21(6):2178–2185 2185
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Supplemental material Systems Pharmacology Approach to Prevent Retinal Degeneration in Stargardt Disease Yu Chen, Grazyna Palczewska, Debarshi Mustafi, Marcin Golczak, Zhiqian Dong, Osamu Sawada, Tadao Maeda, Akiko Maeda, and Krzysztof Palczewski Table of Contents: 1. Supplemental Table 1. 2. Supplemental Table 2. 3. Supplemental Table 3. 4. References. Sup

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