B

Figure 6.2. A) Expression vectors for sense and full and short antisense SERT. B) DNA construct expression in culture. Five ^g of the sense SERT construct was transfected into LLC-PK1 cells ± 25 ^g of each antisense construct. 2.5 ^g of a pRc-CMV DAT was added in each transfection in order to assess its yield. [3H]-5-HT and [3H]-DA uptakes were measured 2 days posttransfection. Mean ± sem of 8-15 determinations. *P<0.001 compared with the value in the absence of antisense DNA (Student's ¿-test).

Figure 6.2. A) Expression vectors for sense and full and short antisense SERT. B) DNA construct expression in culture. Five ^g of the sense SERT construct was transfected into LLC-PK1 cells ± 25 ^g of each antisense construct. 2.5 ^g of a pRc-CMV DAT was added in each transfection in order to assess its yield. [3H]-5-HT and [3H]-DA uptakes were measured 2 days posttransfection. Mean ± sem of 8-15 determinations. *P<0.001 compared with the value in the absence of antisense DNA (Student's ¿-test).

To correct for variations in transfection yields, 2.5 ^g of pRc-CMV containing the entire coding sequence of the DAT gene (Martres et al., 1998) was cotransfected with the various SERT plasmids. The uptake of [3H]DA (46 Ci/mmol, Amersham) was measured as already described with 3-6 nM [3H]DA, in the absence or the presence of 10 ^M nomifensine (RBI) (Research Biochemicals, Natick, MA, USA) to determine nonspecific uptake.

Figure 6.2. C) Complexation of DNA by PEI. SERT DNA construct (0.5 ^g) was mixed with PEI at various charge ratios and complexes electrophoresed in 1% agarose gel stained with ethidium bromide. When the DNA is not entirely neutralized by PEI, the plasmid migrates under coiled (C) and supercoiled (SC) forms. D) Autoradiographic labeling of SERT in the dorsal raphe nucleus 3 days post-injection of sense (sense) or nonrecombinant plasmid (control) or 7 days after administration of the short antisense plasmid (antisense). Increasing OD values corresponded from blue, green, yellow to red. From Fabre, et al., 2000b, copyright 2000, Society for Neuroscience. (See color insert.)

Figure 6.2. C) Complexation of DNA by PEI. SERT DNA construct (0.5 ^g) was mixed with PEI at various charge ratios and complexes electrophoresed in 1% agarose gel stained with ethidium bromide. When the DNA is not entirely neutralized by PEI, the plasmid migrates under coiled (C) and supercoiled (SC) forms. D) Autoradiographic labeling of SERT in the dorsal raphe nucleus 3 days post-injection of sense (sense) or nonrecombinant plasmid (control) or 7 days after administration of the short antisense plasmid (antisense). Increasing OD values corresponded from blue, green, yellow to red. From Fabre, et al., 2000b, copyright 2000, Society for Neuroscience. (See color insert.)

Intra-Cerebral Gene Transfer and Dissection

Adult male Sprague Dawley rats (250-300 g weight) were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and positioned in a stereotaxic apparatus (David Kopf Instruments, Phymap, Paris, France). The needle (outer diameter: 0.46 mm) of a 10 ^l Hamilton syringe was lowered into the DRN (angle 32°C, A = 0.12 cm, L = 0.40 cm, H = 0.28 cm from the interaural zero, according to the atlas of Paxinos and Watson, 1986). Just before injection, plasmid DNA was diluted in 5% glucose at 0.25 ^g/^l. Linearized PEI of 22 kDa (a gift of J.P. Behr) was diluted to 0.01 M in 5% glucose and added to the DNA solu-

tion at 6 charge equivalents. The mixture was vortexed (30 s) and equilibrated for 10 min at RT before injection. Two min after the needled was lowered, 2 |l of the DNA/PEI complex was injected manually over 5 min. The needle was left in place for another 5 min to allow a good diffusion before its removal. Paired control rats received the nonrecombi-nant pRc-CMV under the same conditions of complexation with PEI.

At various times after injection, rats were decapitated and their brains were removed. The hypothalamus was dissected out, and a coronal cut was made at an interaural anteriority ~0.50 cm. The posterior part of the brain was frozen in isopentane at -30°C, and the hippocampus, the striata, and the anterior cortex were dissected out for binding experiments. For immunoautoradiographic experiments, rats were anaesthetized with pentobarbital (60 mg/kg, i.p.) and perfused transcardially with 300 ml of 0.9% NaCl and 0.1% NaNO2. After decapitation, the brain was removed and frozen as described above.

Quantitative Autoradiography

[3H]Citalopram binding: Coronal sections (20 |im) at the level of the DRN were cut at -20°C, thaw-mounted onto gelatin-coated slides, and stored at -20°C until use. For the labeling procedure, slides were brought to RT and preincubated for 15 min in 50 mM Tris-HCl buffer containing 120 mM NaCl and 5 mM KCl, pH 7.4, at 25°C. Incubations proceeded for 1 h at 25°C in fresh buffer with 0.7 nM [3H]citalopram (85 Ci/mmol, Dupont-NEN, Boston, MA, USA). Nonspecific binding was estimated from adjacent sections with 10 |M fluoxetine. Sections were washed twice (5 min) in ice-cold Tris buffer, dried in air, and apposed to 3H-Hyperfilm (Amersham: 4 days, 4°C). Optical density was measured by using a computerized image analysis system (Biocom, les Ulis, France) and converted to femto-moles [3H]citalopram specifically bound per milligram tissue according to a 3H-standard scale.

Immunoautoradiography with anti-SERT, anti-DAT, or anti-5-HT1A receptor antibodies: Polyclonal antibodies specific to the rat 5-HT1Areceptor were used according to Gérard et al. (1994), as well as polyclonal antibodies specific to the rat DAT (Martres et al., 1998) or to the rat SERT (gift of Zhou et al., 1996). Coronal sections (20 |im) were fixed for 3 min with 4% paraformaldehyde in phosphate buffered saline (PBS) (50 mM NaH2PO4\Na2HPO4, 154 mM NaCl, pH 7.4) at 4°C and washed. They were preincubated for 1 h in PBS with 3% bovine serum albumin (BSA) and 1% donkey serum and incubated overnight at 4°C with affinity-purified anti-5-HT1A receptor, nonpurified anti-DAT or anti-SERT antibodies at 1/5000, 1/20,000 or 1/5000 dilution, respectively. After extensive washes, sections were incubated (2 h at RT) in the same solution supplemented with donkey anti-rabbit [125I]IgG (750-3000 Ci/mmol, Amersham) and 1 mM NaI, washed, dried, and apposed to Bmax films (Amersham) for 2-5 days. Optical density on the im-munoradiograms was measured as already described.

[35S]GTP-y-S binding: Autoradiography of agonist-stimulated [35S]GTP-y-S binding was performed as described (Waeber and Moskowitz, 1997). Brain sections (20 |im) were preincubated at 25°C for 15 min in 50 mM HEPES, pH 7.5, containing 100 mM NaCl, 3 mM MgCl2, 0.2 mM ethyleneglycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 0.2 mM dithiothreitol and incubated for 15 min in the same buffer supplemented with 2 mM iguanosine diphosphate GDP (Boehringer Mannheim, Mannheim, Germany) and 10 |M of 8-cyclopentyl-1,3-dipropylxanthine (CPDPX; Research Bio-chemicals), an adenosine receptor antagonist, to reduce nonspecific binding (Laitinen and Jokinen, 1998). Sections were incubated (1 h, 30°C) in the same buffer with 0.05 nM

[35S]GTP-y-S (1000 Ci/mmol, Amersham) supplemented (stimulated conditions) or not (basal conditions) with 10 pM 5-carboxamido-tryptamine (5-CT; Research Biochemi-cals). Nonspecific binding was determined on adjacent sections with 10 pM WAY 100635 (Wyeth-Ayerst, Princeton, NJ, USA). Optical density was measured as above.

[3H]5-HT Synaptosomal Uptake

Brain tissues were homogenized in 15-20 volumes (v/w) of 0.32 M sucrose with a glassTeflon potter (Heidolf Bioblock, Illkizch, France). Homogenates were diluted to 40 vol with 0.32 M sucrose, centrifuged (1000 g, 10 min), and the supernatants were re-cen-trifuged (10,000 g, 30 min). Pellets were gently resuspended in 50 vol of 0.32 M sucrose, and 25 pl aliquots were incubated in 500 pl of uptake buffer (see above) containing 3-6 nM [3H]5-HT, with or without 10 pM fluoxetine to determine nonspecific uptake. After 7 min at 37°C, samples were diluted with 3 ml of ice-cold buffer and rapidly filtered through Whatman GF/B glass-fiber filters (Ihymep) presoaked with 0.05% PEI. After three washes with ice-cold buffer, filters were dried and radioactivity counted by scintillation spectrometry.

[3H]Citalopram Membrane Binding

Tissues were homogenized in 40 volumes (v/w) of ice-cold 50 mM Tris-HCl containing 120 mM NaCl and 5 mM KCl, pH 7.4, by using a Polytron (Touzart-Matignon, Vitzy-sur-Seine, France). Homogenates were centrifuged (40,000 g, 20 min), and pellets were re-suspended in 40 vol of buffer and incubated (37°C, 10 min) to remove endogenous 5-HT. Membranes were spun down (40,000 g, 20 min) and washed threefold by resuspension/centrifugation. The final pellet was suspended in 10 vol of the same buffer, and aliquots (25 pl) were incubated in 500 pl with 0.7 nM or various concentrations (0.2-6 nM) of [3H]citalopram. Nonspecific binding was determined in the presence of 10 pM fluoxetine. After 1 h at 25°C, samples were diluted and rapidly filtered as already described. Kinetic parameters of [3H]citalopram-specific binding were calculated by using the program Ligand.

Measurements of Tissue Levels of 5-HT and its Metabolite

Aliquots (200 pl) of homogenates in 0.32 M sucrose (prepared for the uptake measurement) were mixed with HClO4 (0.1 M final), 0.05% Na2S2O5, and 0.05% disodium EDTA and were centrifuged (30,000 g, 15 min, 4°C). Supernatants were neutralized with 2 M KH2PO4/K2HPO4, pH 7.4, supplemented with 0.01 mg/ml ascorbate oxidase, and KClO4 precipitate was removed by centrifugation (30,000 g, 10 min, 4°C). Aliquots (10 pl) of supernatants were injected directly into a high-performance liquid chromatography (HPLC) column (Ultrasphere IP, Beckman, Gagny, France; 25 cm x 4.6 cm, C18 reversephase, particle size 5 pm) protected with a Brownlee precolumn. The mobile phase (at a 1 ml/min flow rate) consisted of 70 mM KH2PO4, 2.1 mM triethylamine, 0.1 mM disodium EDTA, 1.25 mM octane sulfonate, and 16% methanol, adjusted to pH 3.02 with solid citric acid. The electrochemical detection system (ESA 5011, Collaborative Research, Bedford, MA, USA) comprises an analytical cell with dual coulometric monitoring electrodes (+50 mV and +350 mV). The generated signal was integrated by a computing integrator

(System-Gold, Beckman), and quantitative determinations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were performed with reference to pure standards.

Protein Concentrations

Proteins were quantified according to Lowry et al. (1951) with BSA as standard. Electrophysiological Studies

Young male rats (~100 g weight) were used because brain tissues from such young animals were more resistant to hypoxia occurring for the preparation of brain slices. Rats were decapitated 8 days after intra-DRN injection. Brains were removed rapidly and placed in an ice-cold Krebs solution continuously bubbled with carbogen (95% O2/5% CO2). Vibratome slices (400 pm) containing the DRN were cut (4°C) and maintained at RT in an artificial cerebrospinal fluid for 1 h and transferred to a recording chamber. Extracellular recordings were made with glass microelectrodes (filled with 2 M NaCl, impedance: 10-15 Mft) introduced into the DRN area, as described (Haj-Dahmane et al., 1991). After identification of the cells as serotonergic neurons, basal activity was recorded before application of the 5-HT1A receptor agonist, ipsapirone, at 10-1000 nM. This drug was added to the superfusing fluid for 3 min, and the resulting changes in firing rate of the recorded neurons were computed and registered graphically.

Recording of Sleep/Wakefulness Cycles

Immediately after injection into the DRN of adult male rats with DNA/PEI complexes, animals were implanted with three sets of electrodes (made of enameled nichrome wire, 150 pm in diameter) for polygraphic sleep monitoring (Maudhuit et al., 1994). Two electrodes were inserted through the skull onto the dura over the right frontal and occipital cortex to record the electroencephalogram (EEG), two electrodes were positioned subcu-taneously on each side of the orbit for the electrooculogram (EOG), and two electrodes were inserted into the neck muscles for the electromyogram (EMG). All electrodes were anchored to the skull and soldered to a mini-connector and both were embedded with acrylic cement. Animals were housed in individual recording cages and allowed to recover for 6 days. They were connected to the recording cables for a 2-day habituation. On the eighth day after injection of the plasmids, polygraphic recordings were scored manually every 30 s epoch and, for each animal, the amounts of wakefulness (W), slow wave sleep (SWS), and paradoxal or rapid eye movement sleep (PS or REMS) were computed and calculated over 3-h periods throughout 24 consecutive hours.

Rats were then decapitated and their brains were removed. The hypothalamus was dissected out for the measurement of [3H]5-HT synaptosomal uptake, and the rest of the brain was frozen in isopentane at -30°C for binding and autoradiography.

Statistical Calculations

ANOVA was applied and Student's t-test was used to compare values for rats injected with recombinant constructs with those for paired control rats, injected with the nonre-combinant pRc-CMV.

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