European Journal of Neuroscience, Vol. 19, pp. 855±862, 2004

ß Federation of European Neuroscience Societies

Distinct subcellular targeting of ¯uorescent nicotinic a3b4 and serotoninergic 5-HT3A receptors in hippocampal neurons ReÂgis Grailhe,1 Lia Prado de Carvalho,2 Yoav Paas,1 Chantal Le Poupon,1 Martine Soudant,1 Piotr Bregestovski,3 Jean-Pierre Changeux1 and Pierre-Jean Corringer1 1

ReÂcepteurs et Cognition, Unite de recherche associeÂe D1284, CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France Unite UMR7102 Neurobiologie des processus adaptatifs, Universite Pierre et Marie Curie, Paris, France 3 Institut de Neurobiologie de la MeÂditerraneÂe, INSERM U29, Marseille, France 2

Keywords: GFP, nicotinic, receptors, serotoninergic, targeting

Abstract The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 serotonin receptor subtype belong to a superfamily of neurotransmittergated ion channels involved in fast synaptic communication throughout the nervous system. Their traf®cking to the neuron plasmalemma, as well as their targeting to speci®c subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N-termini of a3b4-nAChR subunits and the 5-HT3AR subunit with cyan and yellow ¯uorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric a3CFP-b4 and YFP-a3b4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP-5-HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP-5-HT3A receptor was found to be targeted to the micropodia in HEK-293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied speci®c ¯uorescent probes. The ef®cient targeting of the YFP-5-HT3A to the cytoplasmic membrane is in line with the large serotonin-elicited currents (nA range) measured by whole-cell voltage-clamp recordings in transfected HEK-293 cells. In contrast, a3b4-nAChRs expressed in the same cells yielded weaker ACh-evoked responses. Taken together, the ¯uorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of a3b4-nACh receptors and the predominant expression of the 5-HT3AR in dendritic surface loci.

Introduction The superfamily of pentameric ligand-gated ion channels consists of the cationic nAChRs and 5-HT3 receptors, as well as the anionic glycine, GABAA/C and GluCl receptors (Karlin & Akabas, 1995; Le NoveÁre & Changeux, 1995; Ortells & Lunt, 1995). These receptor channels mediate and modulate chemical interneuronal communication in the central (CNS) and the peripheral nervous system. To ful®l their physiological role, these proteins are ef®ciently exported to the cytoplasmic membrane and targeted to speci®c subcellular compartments. There is, at the present time, little knowledge of the intracellular traf®cking and targeting of these transmembrane receptors (Williams et al., 1998). The 5-HT3A and a3b4-nACh receptors are organized as functional homopentamers and heteropentamers, respectively. Their transmembrane (TM) subunit topology is dictated by a large N-terminal extracellular domain followed by four transmembrane segments, and a large cytoplasmic region which connects TM3 and TM4 (Fig. 1B). High densities of 5-HT3A binding sites are found in the central nervous

Correspondence: Dr ReÂgis Grailhe or Professor Jean Pierre Changeux, as above. E-mail: [email protected] or [email protected] Received 9 June 2003, revised 6 November 2003, accepted 17 November 2003 doi:10.1111/j.1460-9568.2004.03153.x

system (Barnes et al., 1990; Gehlert et al., 1991), whereas the a3b4nAChRs are mainly distributed in the periphery, where they contribute to synaptic input in autonomic ganglia (Stollberg & Berg, 1987). The availability of the green ¯uorescent protein (GFP), initially cloned from the jelly®sh Aequorea Victoria (Prasher et al., 1992; Lippincott-schwartz & Patterson, 2003), has enabled visualization of a few receptor channel±GFP fusions (Conor et al., 1998; David-Watine et al., 1999; Kittler et al., 2000; Genler et al., 2001; Palma et al., 2002). Here, ¯uorescent protein (FP) variants such as the cyan ¯uorescent protein (CFP) and the yellow ¯uorescent protein (YFP), known to have distinct spectral properties, were fused to the N-terminus of the 5HT3A and a3b4-nACh subunits. Fusion at this position did not impair the receptor-channel activity and allowed us to follow the location of the receptors inside the cell and at the cell surface. Furthermore, we could probe the receptors' topological orientation across the plasma membrane with antibodies directed against the FP, when the receptor was folded and assembled correctly. A striking difference between the surface expression of a3b4nAChRs and 5-HT3A receptors was found. In transfected hippocampal neurons, the pattern of expression shared some similarity with HEK293 cells. In both cell types, most of the nicotinic receptors were found intracellularly rather than at the plasmalemma. In contrast, the 5-HT3A receptors were detected mainly on the cell surface in micropodia of

856 R. Grailhe et al. HEK-293 cell and dendritic spines of hippocampal neurons, with the correct folding and assembly as indicated by electrophysiological and ¯uorescent studies in living cells.

Materials and methods DNA constructs and transfection of HEK-293 cells The ¯uorescent-tagged nicotinic a3 and b4, and serotoninergic 5-HT3A subunits were generated using a strategy analogous to that previously reported for the zebra®sh glycine subunit (GlyRz1) (DavidWatine et al., 1999). Rat a3b4-nicotinic subunits and mouse 5-HT3A long splicing form sequences were ampli®ed by polymerase chain reaction (PCR), from the codon encoding the ®rst amino acid to the stop codon. The resulting PCR products were digested with proper restriction enzymes and ligated into a pMT3-based vector (Swick et al., 1992) containing, in frame, the peptide signal from the GlyRz1, followed by the enhanced cyan or yellow ¯uorescent proteins (CFP, YFP; Ozyme, France). All expression plasmid constructs were veri®ed by restriction mapping and nucleotide sequencing. Endoplasmic reticulum (ER) was labelled by cotransfecting the subcellular expression vector pECFP-ER (Ozyme, France). Cells were transiently transfected on 0.18-mm-thick glass-bottomed dishes (MatTek, USA) precoated with 0.5 mg/mL poly(L-lysine) (Sigma). HEK-293 cell transfection of the constructions was carried out using the LipofectAMINE Reagent PLUS procedure (Gibco BRL) and visualized 2±7 days afterward.

Confocal microscopy A Zeiss LSM510 laser scanning confocal microscope was used (Zeiss, Oberkochen, Germany). Detection and distinction between ¯uorescent signals were achieved by using appropriate conditions (for excitation, emission and beam splitter, respectively): CFP (laser light at 458 nm, BP 475±525 nm, 458/514 nm), YFP (laser light at 514 nm, BP 530± 600 nm, 458/514 nm) and Texas Red±Rhodamine (laser light at 543 nm, HP 585 nm, 488/543 nm). Additionally, to improve resolution and signal-to-noise ratio, images were restored using the Huygens software 2.3.4a (Scienti®c Volume Imaging, Netherlands) and visualized using Imaris 3.1 software (Bitplane, Switzerland). Calculations were performed on a Silicon Graphics Octane workstation. Quantitative image processing and analysis of optical sections were performed with ImageJ software (http://rsb.info.nih.gov/ij/). Immunolabelling of HEK-293 cells Transiently transfected HEK-293 cells attached to glass-bottomed dishes were rinsed with PBS (3  5 min) and then ®xed without permeabilization in 4% paraformaldehyde solution for 15 min at 4 8C. After several washes with PBS, the cells were incubated with monoclonal antibody against the conserved ectodomain of FP (Invitrogen) variants diluted 1 : 5000 in a PBS solution containing 0.1% BSA and 10% horse serum. After three washes in PBS containing BSA 0.1%, they were then incubated for 1 h with the Texas Red-conjugated goat antimouse antibody (Jackson ImmunoResearch).

Electrophysiology

Rat hippocampal primary culture and transfection

Agonist-evoked currents were monitored at room temperature in the whole-cell patch-clamp con®guration using EPC-9 (HEKA Electronics, Lambrecht, Germany), 2±4 days after transfections. The external solution (pH 7.3) contained (in mM): NaCl, 140; CaCl2 2; KCl, 2.8; MgCl2, 2; HEPES/NaOH, 10; and glucose, 10. In experiments with 5-HT3A receptors, MgCl2 and CaCl2 were omitted from the extracellular solution and 1 mM EGTA was added. A pressure-driven system with three parallel square tubes was used to deliver solutions. The tubes were positioned 40±50 mm above the recorded cell and connected to a computer-driven fast exchange system (SF 77 A Perfusion Fast-Step, Warner, USA), allowing a 10±90% solution exchange in 1±5 ms, as measured by open electrode controls (1/10 external solution/water). Whole-cell currents were recorded at holding potentials of 100 mV. The internal solution (pH 7.3) contained (in mM): CsCl, 140; MgATP, 2; HEPES/CsOH, 10; and BAPTA, 10. Series resistances (5±15 MV) were compensated to 85±90%. Current± voltage relationships were determined by two methods; ®rst, agonistevoked currents were recorded at different holding potentials ranging from 100 to ‡50 mV. In the second method, inverted voltage ramps (sweeping from ‡50 to 100 mV in 200 ms) were applied in the absence or in the presence of the agonist (1 s after the beginning of a 3-s application). In this case, the agonist-evoked current was obtained by subtracting leak currents recorded in the absence of agonists.

Hippocampal primary cultures were prepared from 17-day-old rat embryos. The pregnant females were anaesthetized with CO2 and killed by cervical dislocation. Rat embryos were decapitated, and isolated hippocampi were mechanically and chemically dissociated in single-cell suspension with trypsin (0.25 mg/mL) (Brewer et al., 1993). The cells were centrifuged and suspended in Neurobasal medium (GibcoBRL) supplemented with Glutamax I (GibcoBRL), B27 (GibcoBRL), 100 U/mL penicillin and 100 mg/mL streptomycin (DMEM medium; GibcoBRL). The dissociated cells were plated onto 0.18-mm-thick glass-bottomed dishes precoated with 0.5 mg/mL poly(L-ornithine) (Sigma). After 5 days, primary culture neurons were transfected for 4 h using the LipofectAMINE Reagent PLUS method (Gibco BRL), to be observed 2±7 days later. No signi®cant differences in cellular distribution for the ¯uorescent receptors were seen at different times after transfection. Every 4 days, half of the culture medium was replaced with fresh Neurobasal supplemented medium. For neuronal staining, primary neurons were permeabilized in 0.1% Triton in PBS for 5 min immediately after ®xation. The primary antibody used was a mouse monoclonal anti-NeuN antibody (1 : 200; Chemicon, Temecula, CA, USA) which qualitatively stains the NeuN antigen expressed speci®cally by neuronal cells, and the secondary antibody was a goat antimouse IgG antibody, Alexa 568labelled (1 : 500; Molecular Probes). Tracking of transfected neurons was performed at low magni®cation, when ¯uorescent tagged receptors were coexpressed with a spectrally distinct ¯uorescent protein (examples in Figs 5B and 6B; CFP). For 5-HT3A surface receptor staining, we used the antagonist GR-H (GR119566), covalently labelled with Rhodamine B isothiocyanate (GR-rho; Schmid et al., 1998). The primary hippocampal neuron culture was incubated for 5 min in culture medium containing 2 nM GR-rho and then rinsed with Neurobasal medium before image recording.

Digital-imaging fluorescence microscopy Epifluorescence microscopy Fluorescence images were acquired using a Zeiss Axiovert ¯uorescent microscope which was ®tted with a Sensicam charge-coupled device camera (12-bit) and controlled by the MetaVue software package (Universal Imaging Corporation, USA). Filter cube speci®cations for the ¯uorescence channels were as follows for excitation, emission and main dichroic beam splitter, respectively: CFP, 440  21, 480  30 and 455 nm; YFP, 500  25, high-pass ®lter (HP) 530 and 525 nm (Chroma, USA).

Image analysis Analysis of the distribution of the ¯uorescent receptors in the dendritic compartment of hippocampal cells was accomplished as follows. Two

ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 855±862

Targeting of fluorescent nACh and 5-HT3A receptors

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circular zones were set, a circular zone which contained the entire soma (S) and a second surrounding zone which contained the dendritic processes (D). The area of zone D was twice the area of zone S, which was excluded from zone D. The ®nal grey values for the dendritic zone were normalized to the ¯uorescence of its S zone. Quantitative image processing was performed with ImageJ software.

Results Constructions of a3b4-nAChR and 5-HT3AR subunits tagged with fluorescent proteins The cDNAs encoding for the CFP and YFP were introduced as illustrated in Fig. 1. The corresponding ¯uorescently tagged subunits (FP-tagged) are termed YFP-a3, CFP-b4 and YFP-5-HT3A. FP-tagged receptor subunits formed functional channels To ascertain whether the FP-tagged subunits designed here assemble into fully functional receptor channels, their electrophysiological properties were compared to those of nontagged receptors in whole-cell patch-clamp recordings performed in transiently transfected HEK-293 cells. The coexpression of a3b4-nAChR subunits is required to form functional hetero-pentameric channels (Duvoisin et al., 1989). In 50% of the cells transfected with wild-type a3b4- or ¯uorescent YFP-a3b4- and a3CFP-b4-nAChRs, ACh elicited inward currents at a holding potential of 100 mV. Responses were proportional to the concentration of ACh (100±300 mM range) and showed slow desensitization with a similar time course (Fig. 2A±C). Mean currents elicited by 300 mM ACh were 710  228 pA for a3b4 (n ˆ 6), 335  63 pA for a3CFP-b4 (n ˆ 10) and 175  29 pA for YFPa3b4 (n ˆ 6). In all cases, current±voltage relationships displayed the strong inward recti®cation typical of nAChRs (Fig. 2D). Contrary to cells transfected with nAChR, almost 100% of the cells transfected with either wild-type or YFP-tagged 5-HT3A subunits responded to serotonin. In these cells, serotonin (10±100 mM) elicited robust rapidly desensitizing inward currents with an amplitude reaching several nA at 60 mV (Fig. 2E), and similar current±voltage relationships were observed (data not shown). The mean maximal current caused by a saturating concentration of serotonin (100 mM) in

Fig. 2. Functional expression of FP-tagged a3b4-nAChR and 5-HT3AR. Neurotransmitter-evoked currents in HEK-293 cells expressing wild-type and ¯uorescently tagged a3b4 and 5-HT3A receptors: (A) a3b4; (B) a3 is the FP-grafted subunit; (C) b4 is the FP-grafted subunit; and (E) YFP-5-HT3A homopentameric receptor. The horizontal bar indicates the time of ACh or 5-HT applications, at concentrations indicated in the ®gure. Holding potential, 100 mV. (D) Current±voltage relationship of the a3b4 FP-tagged receptor channels; the bold line corresponds to the wild-type a3b4 receptor.

cells transfected with YFP-5-HT3A was 3125  1709 pA (n ˆ 4). These currents were strongly inhibited by calcium or magnesium (2 mM) (data not shown and Maricq et al., 1991; Eisele et al., 1993), as was the case in cells transfected with untagged 5-HT3AR. Our results thus show that the fusion of a FP to the N-terminus of either a3 or b4 nAChR subunits or to the serotonin 5-HT3A subunit generates receptors which can be expressed as fully functional ionic channels, displaying the electrophysiological properties typical of native receptors. The weaker currents elicited by agonists and the lower proportion of transfected cells expressing the a3b4-nAChR compared to 5-HT3AR was characteristic of nicotinic receptors, regardless of the presence or absence of the FP. 5-HT3ARs but not a3b4-nAChRs were largely expressed at the surface of HEK-293 cells Fig. 1. Schematic representation of ¯uorescent subunits. (A) The ¯uorescent protein (FP; dotted box) was inserted between a common leader peptide (LP) and the ®rst amino acid of the mature rat a3b4-nicotinic receptor subunits (white box) and the mouse 5-HT3A receptor subunit (grey box). The numbered black boxes correspond to transmembrane domains. (B) A proposed transmembrane (TM) topology of FP-a3 tagged subunit (left), and a top view of FP-a3b4 pentameric organization (right).

To determine whether the FP-tagged a3b4-nAChR and 5-HT3AR produce detectable ¯uorescence, HEK-293 cells were transiently transfected with various tagged subunits. The coexpression of YFPa3 with wild-type b4 and the coexpression of CFP-b4 with wild-type a3 were investigated. 5-HT3A subunits are known to form functional homo-oligomers, so the YFP-5-HT3A fusion was transfected alone (Maricq et al., 1991).

ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 855±862

858 R. Grailhe et al. Two to seven days after transfection, HEK-293 cells were visualized by epi¯uorescence (Fig. 3A±F) and confocal microscopy (Fig. 3G±L). Nicotinic and serotoninergic receptors displayed distinct ¯uorescence distributions. Thus, for the YFP-a3b4 and a3CFP-b4 receptors, the ¯uorescence was distributed within cytoplasmic compartments of the cell (Fig. 3E). In contrast, expression of YFP-5-HT3AR yielded ¯uorescence both in intracellular compartments and near or within the plasma membrane (see arrows in Fig. 3B). This difference was further revealed by transfecting the same cells with YFP-5-HT3A receptors together with a3CFP-b4 nAChR (Fig. 3G±I); this clearly showed that the yellow ¯uorescence of YFP-5-HT3AR was colocalized with the cyan ¯uorescence of a3CFP-b4 in intracellular compartments, while only yellow ¯uorescence was observed at the plasma membrane (Fig. 3I). To further demonstrate that the YFP-5-HT3AR fusion receptor was present at the cell surface, nonpermeabilized transfected HEK-293 cells were interacted with a speci®c antibody directed against FP. Figure 3K and L show labelling of the cell surface with the antibody±

Fig. 3. The YFP-5-HT3AR, unlike the a3CFP-b4 nAChR, is predominantly expressed at the surface of living HEK-293 cells. (A±F) epi¯uorescence; (G±L) confocal microscopy. (A±C) HEK-293 cells expressing the YFP-5-HT3A receptor. (D±F) HEK-293 cells expressing the a3CFP-b4 receptor. (A,D) Transmission image; (B,E) epi¯uorescence; (C) A and B merged; (F) D and E merged. The YFP-5-HT3A ¯uorescent signal appears to be localized to the cytoplasmic membrane (B; see arrows). Blue and yellow correspond to CFP and YFP, respectively. (G±I) Co-expression of a3CFP-b4 and YFP-5-HT3A receptors in HEK-293 cell (I, merge of G and H) shows (G) intracellular and (H) intracellular plus plasmalemma ¯uorescence labelling. (J±L) Non-permeabilized HEK-293 cells expressing YFP-5-HT3A (J) were incubated with anti-FP antibodies and detected with secondary antibodies conjugated to Texas Red (TxRed anti-FP; K). The orange labelling seen in panel L at the cell surface corresponds to colocalized ¯uorescence (merged J and K). Scale bar, 10 mm (A±F), 5 mm (G±L).

Texas Red conjugates, indicating the presence of the YFP-5-HT3A receptors on the cell surface. We found that the ¯uorescent cells expressing the YFP-5-HT3A receptors presented some speci®c labelling for the FP at the cell surface. For a3CFP-b4 and YFP-a3b4nAChRs, no substantial surface labelling could be detected in 1000 ¯uorescent HEK-293 cells expressing FP-a3b4 receptors, in three independent experiments (data not shown). We additionally looked for expression of a3b4 at the cell surface with pharmacological tools. For this purpose, we used [3H]-epibatidine, a ligand which, unlike carbamylcholine, crosses cell membranes (Whiteaker et al., 1998). We found labelling of receptors but no substantial displacement of [3H]epibatidine with carbamylcholine (data not shown), further indicating that the large majority of a3b4 receptors are expressed intracellularly. One advantage of live imaging is that it enables visualization of the microextensions of the plasma membrane that are otherwise easily damaged by ®xation procedures or mechanical force during specimen preparation. Indeed, more detailed analysis of these images showed that the 5-HT3AR is speci®cally targeted to the tips of micropodia (Fig. 4H). 3-D reconstruction of a stack of confocal optical sections of HEK-293 cells coexpressing YFP-5-HT3AR with endoplasmic reticulum (ER) protein marker (ER-CFP) revealed that the receptor was located in the plasmalemma and the micropodia (Fig. 4J).

Fig. 4. Subcellular localization of YFP-a3b4 and YFP-5-HT3A receptors in HEK-293 cells. (A±C) Expression of (A) ER-CFP marker and (B) YFP-a3b4 receptor in the same HEK-293 cell; (C) A and B merged image. (D±F) Expression of (D) ER-CFP marker and (E) YFP-5-HT3A receptors in the same cell; (F) D and E merged. Intracellular and cell surface expression is detected in panels E, F and J. Regions shown in rectangles in D±F are enlarged in G±I, respectively. (J) 3-D reconstruction view of HEK-293 cell coexpressing YFP-5HT3AR and ER-CFP as generated by a confocal stack of 140 optical sections spaced at 100 nm. Note that several micropodia display strong labelling (H and I, arrows). Scale bars, 10 mm (A±F), 2 mm (G±I), 3 mm (J).

ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 855±862

Targeting of fluorescent nACh and 5-HT3A receptors

Fig. 5. Visualization of a3b4-nAChR and 5-HT3AR in a single hippocampal neuron. (A±D) Triple-labelled hippocampal neurons (D) show the colocalization of (A) YFP-5-HT3A, (B) CFP and (C) the neuronal marker NeuN. (E,F) Living hippocampal neuron coexpressing (E) CFP-a3b4 and (F) YFP-5-HT3A receptors shows differences in receptor localization (in which pixel intensities have been inverted for clarity; see insets). The dotted white lines in panel E (cyan) and F (yellow) correspond to sections of soma (line 1) and dendrites (line 2) in which pixel intensities for each channel were quanti®ed (panel G). The ¯uorescence intensity values for YFP-5-HT3AR (yellow line) reveal distinct intensity peaks corresponding to dendrite compartments, while CFP-a3b4 ¯uorescence (cyan line) is present in the cell body. The arrows in the inset in panel F point to preferential targeting of 5-HT3AR. (H) Neurons coexpressing both receptors (n ˆ 18) were subdivided into two areas, the soma (S) and the dendritic area (D). Total ¯uorescence intensity in the dendritic compartment was normalized to the level of the soma for CFP-a3b4 (cyan) and YFP-5HT3AR (yellow); arbitrary units (A.U.) Scale bars, 30 mm (A±D); 15 mm (E,F).

Intracellular a3b4-nAChRs and 5-HT3ARs were located in the ER To further elucidate the distribution of receptors in cellular compartments, YFP-tagged subunits were cotransfected with the ER ¯uorescent marker ER-CFP. YFP-a3b4 expressed in HEK-293 cells along with ER-CFP protein marker showed a strong colocalization of the two labels (Fig. 4A±C), indicating that the a3b4 receptor was mainly located in the ER. YFP-5-HT3AR was found in intracellular vesicles, and to a lesser extent in the ER (Fig. 4D±J). Time imaging sequences of the vesicles expressing the YFP-5-HT3AR followed linear trajectories, consistent with a microtubule-mediated movement (data not shown). 5-HT3AR but not a3b4-nAChR was efficiently targeted to the plasmalemma in hippocampal neurons Transfected neurons in the primary hippocampal culture were identi®ed with a neuronal-speci®c antibody directed against the neuronal marker NeuN (Mullen et al., 1992; see also Materials and methods

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Fig. 6. The 5-HT3AR is targeted to the cell surface of living hippocampal neurons. Living hippocampal neurons expressing both (A) YFP-5-HT3A receptors and (B) CFP. The regions delineated by rectangles in A, B, D and F are magni®ed in D, C, E and F, respectively. YFP-5-HT3A receptors (yellow) are localized in micropodia end points (C; see arrows) and spine-like formations (E; see arrows). Panels F and G show the cell surface labelled with rhodaminylated competitive antagonist speci®c for the 5-HT3AR (GR-rho; see Material and Methods). By combining triple ¯uorescence we could view in the same neuron the YFP-5-HT3A receptor (yellow in A, C, D and E) and surface-located receptors (yellow in F and G). CFP (cyan in B±G) is homogenously distributed inside the cell (B) and is readily visualized inside dendritic processes (D±G). Note that the YFP-5-HT3A receptor expression pattern (D,E) matches GR-rho ligand labelling (F,G). Scale bars, 30 mm (A,B), 5 mm (C,E,G), 15 mm (D,F).

section). As illustrated in Fig. 5A±D, antibody staining revealed that the transfected neurons expressed a fair amount of ¯uorescent tagged receptors. In order to examine the neuronal distribution of 5-HT3AR and a3b4-nAChR, primary cultures of rat hippocampal neurons were cotransfected with YFP-5-HT3A ‡ a3CFP-b4 or with YFP-5HT3A ‡ CFP. Confocal microscopy showed that the YFP-5-HT3A and a3CFP-b4 receptors were located in a somatic compartment colocalizing with the ER marker (data not shown). Nevertheless, differences in receptor targeting were revealed by expressing a3CFP-b4 nAChRs and YFP-5-HT3ARs in the same cells (Fig. 5E and F). To take into account differences in the level of protein expression and imaging parameters, the ¯uorescence signals of a3CFP-b4 receptors (Fig. 5E) and YFP-5-HT3A receptors (Fig. 5F) were normalized with respect to each other by matching the ¯uorescence in the cell body region. Two-channel quantitative analysis of normalized ¯uorescent intensities indicated that, unlike a3CFP-b4 which remained in the cell body, YFP-5-HT3A was highly localized in the dendrites (Fig. 5G). Additionally, in order to further analyse the distribution of the ¯uorescent receptors, we have developed a methodology which quanti®es the ¯uorescence intensity in the dendritic

ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 855±862

860 R. Grailhe et al. compartment and normalizes by matching the ¯uorescence intensity of the cell soma (see Materials and methods). Two circular zones were selected, a ®rst circular zone which contains the entire soma (S) and a second surrounding zone which contains some of the dendritic processes (D, Fig. 5H). Such analysis was repeated on 18 neurons coexpressing a3CFP-b4 nAChRs and YFP-5-HT3ARs. After normalization of the signal, on average there was, in the dendritic region, a signi®cant net higher pixel intensity in the yellow channel (YFP-5-HT3A, 25.1  5.8 grayscale value) by 2.4  0.45-fold than the cyan channel (a3CFP-b4, 13.3  4.7 grayscale value) (Fig. 5H). These results indicate that, in primary hippocampal neurons, the a3CFP-b4 nAChRs compared to the YFP-5-HT3ARs remain more speci®cally in the soma than in the dendritic processes. Closer examination of primary hippocampal neurons transfected with the YFP-5-HT3A receptor revealed punctuate ¯uorescence in dendritic structures in NeuN-positive cells. More speci®cally, the ¯uorescent receptor was targeted to the tips of ®lipodia and spines. To ascertain that 5-HT3AR seen in dendritic structures was facing the extracellular milieu, we used a ¯uorescent ligand that speci®cally binds to the extracellular domain of the 5-HT3AR, the antagonist GR-H (GR119566) covalently attached to Rhodamine B isothiocyanate and here referred to as GR-rho (Schmid et al., 1998). Detailed analysis of the GR-rho ¯uorescence revealed speci®c labelling at the cell body and dendritic structures of the transfected hippocampal neurons (Fig. 6F and G).

Discussion Cationic ligand-gated ion channels are expressed in various CNS structures and play key roles in a diversity of high brain functions, such as attention, memory, reward, nociception and neuronal development (Marubio & Changeux, 2000; Rezvani & Levin, 2001; Mansvelder & McGehee, 2002). The nAChR and 5-HT3R are implicated in these processes by regulating neurotransmitter release and mediating fast synaptic transmission in the brain (Costall et al., 1990; Jones et al., 1999). Understanding the physiological role of receptors requires knowledge of their electrophysiological properties, cellular localization, traf®cking and targeting in living neurons. The aim of the present work was to address such issues in living cells, with the assistance of ¯uorescent proteins which allow real-time image acquisition without the damaging usage of ®xatives, in HEK293 cells and in cultured hippocampal neurons. To date, several ¯uorescently tagged cationic ligand-gated ion channels have been reported in the literature. GFP has been introduced in the g and e muscle nAChR subunits (Genler et al., 2001) and fused in the C terminal portion of the a7 nACh subunit (Palma et al., 2002). However, these ¯uorescent receptors had never been used for the study of receptor targeting in CNS neuronal cells. Distinct cellular targeting of a3b4-nACh and serotoninergic 5-HT3A receptors With ¯uorescence microscopy, we found FP-a3b4 receptors mostly located in intracellular compartments of transfected HEK-293 cells and not visible at their surface, whereas YFP-5-HT3A receptors were ef®ciently expressed at the HEK-293 cell surface. These observations were further supported by electrophysiological recordings. Indeed, based on the amplitude of whole-cell currents and assuming respective conductance of 29 pS for a3b4 (Stetzer et al., 1996) and 0.31 pS for 5HT3A (Brown et al., 1998) receptors, we estimated that a saturating concentration of ACh or 5-HT activated, at the peak of the response, between 80 and 800 FP-a3b4 and 1100±25000 YFP-5-HT3A receptor channels. In the case of the FP-a3b4 receptor channels, this low level

of surface receptors is insuf®cient for detection by immunolabelling techniques. This observation was in agreement with previous binding experiments showing that a3b4 receptors expressed in HEK-293 are accessible to [3H]-epibatidine, an agonist capable of crossing lipid bilayers, but not accessible to carbamylcholine, a nicotinic ligand which cannot cross the cell membrane (data not shown; Whiteaker et al., 1998). Taken together, the results indicate that the large majority of a3b4 binding sites remain inside the cell and are inaccessible from the extracellular milieu. However, some a3b4 receptors did reach the cell surface and responded electrophysiologically to ACh, illustrating the high sensitivity of the electrophysiological assay. Unlike the a3b4-nAChR, a large fraction of the 5-HT5AR was exported to the cytoplasmic membrane with a correct transmembrane orientation dictating an extracellular localization of the FP epitope, as shown in Fig. 3K and L. Furthermore, high-resolution microscopy indicated the presence of clusters in micropodia end points (Fig. 4H and I). These observations were in line with the large whole-cell current responses measured in HEK-293 cells expressing the 5-HT3AR (Fig. 2E). The comparison performed here clearly showed distinct cell surface expression of the two receptor types, and suggested that inherent structural properties of these receptors regulate their compartmentalization. Unlike the a3b4-nAChR, the 5-HT3AR is targeted to dendritic spines in primary culture of hippocampal neurons The expression pattern of the a3b4-nAChRs in transfected hippocampal neurons was similar to that observed in HEK-293 cells. That is, most of the receptors were found in intracellular compartments, inside the cell soma. Very little a3b4 was found in dendritic process. A similar expression pattern has been reported in the case of paraformaldehyde-®xed ciliary ganglion neurons in culture (Stollberg & Berg, 1987). In this system, the majority of neuronal nicotinic receptors containing the a3 and b4 subunits were colocalized inside cells with monoclonal antibody mAb35. Large intracellular pools of receptors were also consistently reported in the case of a4- and b2containing nAChR receptors, in both transfected cell lines (Whiteaker et al., 1998) and brain slices (Hill et al., 1993; Arroyo-JimeÂnez et al., 1999). The FP tag also allowed further localization of the intracellular pool of receptors at the level of the endoplasmic reticulum, where pentameric ligand-gated ion channels are considered to mature before being exported to the cell surface (Merlie & Lindstrom, 1983). For a3b4 receptors, the inef®cient cell surface expression may have been due to an incomplete folding or assembly of the subunits, resulting in protein retention through the quality control process of the cellular machinery (Ellgaard & Helenius, 2001). Alternatively, it is possible that the subunits matured ef®ciently but remained intracellularly `waiting' for a signal that could trigger their transport to the cell surface, as is the case for AMPA-selective ionotropic glutamate receptors (Luscher et al., 1999). Our experiments suggest that at least part of this intracellular pool corresponded to receptors that were folded in a native conformation. Indeed, (i) the generated subunits were ¯uorescent, indicating a correct folding of the FP portion of the molecule, and (ii) a signi®cant fraction of these receptors did bind epibatidine with high af®nity (data not shown), indicating correct subunit±subunit interactions as the agonist binding sites are located at the subunit interfaces (Corringer et al., 2000). The physiological role of intracellular pools of nAChRs in central and peripheral neurons remains to be elucidated. The precise localization and functions of 5-HT3AR within neuronal circuits is still a matter of debate. 5-HT3AR mRNA is strongly

ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 855±862

Targeting of fluorescent nACh and 5-HT3A receptors expressed in the hippocampal formation (Tecott et al., 1993). Immunocytochemical studies previously performed on brain sections reported labelling of 5-HT3A receptors in cell bodies, dendrites and varicose axons of hippocampal native neurons (Spier et al., 1999), with occasional denser spots in the nucleus of the solitary tract, where the receptors appeared to be essentially associated with small-diameter neurites (Doucet et al., 2000). Our results in living neurons clearly showed that, in cultured hippocampal neurons, 5-HT3AR is targeted to ®lopodias as well as spine heads, where it is folded in a topology allowing correct binding of neurotransmitter and signal transduction (Fig. 6). A similar targeting pattern has been previously reported for FP-tagged NMDA-selective ionotropic glutamate receptors (Luo et al., 2002), known to mediate fast synaptic transmission in the mammalian brain. Interestingly, fast excitatory transmission mediated by 5-HT3 receptors has been recorded in the rat lateral nucleus of the amygdala (Sugita et al., 1992).

Acknowledgements We thank Drs R. Hovius and H. Vogel for providing the ¯uorescent 5-HT3A ligand (GR-rho). We are indebted to N. Mechawar for critically reading the manuscript and for insightful discussions. The authors also would like to thank Drs B. David-Watine, S. Bolher, U. Maskos, J. Sallette, H. Tsuneki and S. Garbay for helpful inputs. The technical assistance of Pascal Roux and Dr Spencer Shorte from the Institut Pasteur Centre for Dynamic Imaging is gratefully acknowledged. This work was supported by the ARC foundation and Institut Pasteur Transverse Research Program no. 40.

Abbreviations BP, band-pass ®lter; CFP, cyan ¯uorescent protein; ER, endoplasmic reticulum; FP, ¯uorescent protein; GFP, green ¯uorescent protein; GlyRz1, zebra®sh glycine subunit; GR-rho, Rhodamine B isothiocyanate; HP, high-pass ®lter; LP, leader peptide; PCR, polymerase chain reaction; TM, transmembrane; YFP, yellow ¯uorescent protein.

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