Poster
# 95
Poster |
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6th Internet World Congress for Biomedical Sciences |
Nicola B. Mercuri(1), Pernilla Grillner(2)
(1)IRCCS Santa Lucia, University of Rome Tor Vergata - Rome. Italy
(2)Dept Physiology and Pharmacology. Karolinska Institutet - Stockholm. Sweden
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[Cell Biology & Cytology] |
[Neuroscience] |
Albino Wistar and Sprague-Dawley rats (100-250 g) (Morini - Reggio Emilia, Italy and BK Universal – Sollentuna, Sweden) were anaesthetised with halothane and killed with a blow to the chest or by decapitation. The brain was rapidly removed from the skull and horizontal slices (300 µm) of the ventral mesencephalon were cut using a vibratome. The slices containing the VTA and SNPC were transferred to a recording chamber continuously perfused with Ringer solution (mM): NaCl 126, KCl 2.5, MgCl2 1.2, NaH2PO4 1.2, CaCl2 2.4, glucose 11, NaHCO3 19, warmed to 35 °C and saturated with 95 % O2, 5 % CO2 (pH = 7.4). The SNPC and VTA were identified as the region medial (VTA), rostral and caudal (SNPC) to the medial terminal nucleus of the accessory optic tract.
Intracellular recordings were performed using thick wall borosilicate glass electrodes (Clark), pulled on a Brown-Flaming (Sutter Instruments) electrode puller. The electrodes were filled with KAc (2 M) or KCl (2M) and had a resistance of 60-110 MW. The signal was obtained with an amplifier (Axoclamp 2A, Axon instrument) in bridge mode and displayed, averaged and stored on a computer using the pClamp software (Axon Instruments). The membrane potential was recorded at resting membrane potential when the spontaneous firing activity was studied, whereas negative current was injected to suppress firing activity when synaptic potentials were recorded.
Individual neurones of the SNPC and VTA were visualised using infrared video microscopy (Hamamatsu, Japan). Recording electrodes were pulled (3–5 M using a vertical puller PP-83 (Narishige, Japan) and filled with a solution containing (mM): KCl 120; MgCl2 2; CaCl2 1; EGTA 11; HEPES 10 (pH 7.3, with KOH). In experiments aiming at the study of glutamatergic synaptic responses, K-gluconate (130 mM) was used instead of KCl. Membrane current and potential were monitored using an Axopatch 1D patch clamp amplifier (Axon Instruments). Usually neurones were recorded at a holding potential of -60 mV. Series resistance ranged from 10 to 20 M and was not compensated in order to maintain the highest possible signal-to-noise ratio. However, cells where series resistance changed by more than 10% during drug application were discarded from the analysis. Data were filtered at 1 kHz, digitised at 10 kHz with a Digidata 1200 hardware and acquired with pClamp software (Axon Instruments).
The postsynaptic potentials or currents (PSP/Cs) were evoked by a local electrical stimulus (0.03 ms, 10-25 V), delivered at a frequency of 0.1-0.3 Hz and generated by a stimulator (Grass Instruments) through a bipolar tungsten electrode placed close to the recording site in the VTA and the SNPC. The stimulus intensity was adjusted subthreshold for action potential initiation. To evoke slow excitatory PSP/Cs (EPSP/Cs) or inhibitory PSP/Cs (IPSP/Cs) short trains (40-400ms, 100-300 Hz) of repetitive stimulating pulses were used. Fast EPSP/Cs were pharmacologically isolated by the bath application of picrotoxin (PTX; 100 µM) or bicuculline methiodide (30 µM) to block the fast GABAA-mediated IPSP/C. During the recording of fast IPSP/Cs, CNQX (10 µM) and AP5 (50 µM) were present to block the EPSP/C and in some cases also the GABAB antagonist saclofen (200 µM) to block the slow IPSP/C. Recordings of the slow IPSP/C were performed in the presence of PTX (100 µM) or bicuculline methiodide (30 µM), CNQX (10 µM) and AP5 (50 µM).
The peak amplitude of the PSP/Cs was measured on averages of 4-10 traces in each different experimental condition. The peak baseline was measured as the average membrane potential during the 100 ms preceding the stimulus artefact. The peak amplitude was defined as the maximal amplitude difference from baseline following the stimulus artefact. Numerical data were expressed as mean ± S.E.M. Statistical differences were evaluated by paired t-test.
Spontaneous synaptic currents were detected using the Mini Analysis Program (Synaptosoft Inc., USA). The cumulative amplitude and interval distributions of the miniature IPSCs (mIPSCs) or spontaneous EPSCs (sEPSCs) were compared using the non-parametric Kolmogorov-Smirnov test.
Drugs were made in stock solutions and bath applied at known concentrations via a three-way tap system. A complete exchange of the solution in the recording chamber occurred in about 1 min. To avoid desensitisation, nicotine was also applied by local pressure by using a glass pipette containing nicotine with the tip placed close to the recording site connected to a Picosprizer II (General Valve Corporation).
The following substances were used: 4-aminopyridine (4-AP), atropine sulphate, bicuculline methiodide, carbachol, dicyclomine, dopamine hydrochloride, glutamate, ketamine, mecamylamine, muscarine chloride, N-ethylmaleimide (NEM), nicotine di-(+)-tartrate, physostigmine, picrotoxin, tetrodotoxin (TTX) (all from Sigma), 2-amino-5-phosphonopentanoic acid (AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), dihydro-b-erythroidine (DHBE), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), (4-hydroxy-2-butynyl)-1-trimethanylammonium-m-chloro-carbanilate chloride (McN-A-343), methoctramine tetrahydrochloride, methyllycaconitine (MLA), pirenzepine dihydrochloride, saclofen (all from RBI), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD), (S)-2-amino-2-methyl-4-phosphonobutanoic acid/(a-methyl-AP4) (MAP4), L-2-amino-4-phos-phonobutyric acid (L-AP4), (RS)-4-carboxy-3-hydroxyphenylglycine (4C3HPG), (2S,1’S,2’S)-2-(carboxycyclopropyl)glycine (L-CCGI), (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG), (±)-a-methyl-4-carboxyphenylglycine (MCPG), L-serine-O-phosphono-butanoate (L-SOP) (all from Tocris Cookson), w-conotoxin GVIA (w-CgTx GVIA), w-agatoxin IVA (w-AgTx), w-conotoxin MVIIC (w-CgTx MVIIC) (all from Almone Labs), CGP35348 (Ciba-Geigy), Isradipine (Sandoz), Nimodipine and Bay-K 8644 (Bayer).
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[Cell Biology & Cytology] |
[Neuroscience] |
