Cholinergic magnocellular basal forebrain neurones were grown in dissociated culture with other basal forebrain cell types using the method o f Allen, Sim & Brown (1993).
2.1.1 Dissociation procedure
Post-natal day 11-14 Sprague-Dawley rat pups were killed by decapitation (no anaesthesia). The brain was rapidly removed and placed in ice-cold Gey’s balanced salt solution supplemented with 0.6% (w/v) D-glucose (total 0.7%) and 8mM MgCl2 (total 8.28mM) hereafter referred to as GBSS. After
hemisection in GBSS, 4(X)/xm coronal slices were cut using a Mcllwain tissue chopper and the slices returned to GBSS. Regions of the basal forebrain nuclei within the slices were visually identified using a light microscope
(transillumination, approx. x2 0 total magnification).
Using the classification of Mesulam et al as recounted by Wainer & Mesulam (1990), regions corresponding to Ch3 (nucleus of the horizontal limb of the diagonal band of Broca) and Ch4 (substantia innominata/nucleus basalis of Meynert) were dissected from the slices. The tissue fragments were washed
twice in Ca^"^-, Mg^^-free Hanks’ balanced salt solution containing lOmM HEPES (HEPES/HBSS) then once in a similar solution containing 0.125 % (w/v) trypsin. The tissue fragments were then resuspended in trypsin-containing HEPES/HBSS and incubated for 1 hour at 37°C in an humidifed atmosphere of 5 % CO2 in air, with one change into fresh trypsin-containing medium after 30
minutes.
After enzyme treatment, tissue fragments were washed repeatedly in Ca^'^-, Mg^^-free Hanks’ balance salt solution supplemented with 8mM MgCl2,
10% (v/v) heat-inactivated foetal calf serum and 10/xg/ml deoxyribonuclease I. The tissue was then transferred to a similar solution containing 20/ig/ml deoxyribonuclease I and mechanically dissociated by trituration through a flame-polished pasteur pipette.
The resulting cell suspension was centrifuged at 27g for 10 minutes and the pellet resuspended in approximately 1ml of growth medium o f the following
composition:
40% (v/v) Dulbecco’s modified Eagle’s medium 40% (v/v) Leibovitz’s L-15 medium
1 0% (v/v) foetal calf serum
25ng/ml Nerve Growth Factor 7S 0.29mg/ml L-Glutamine
6 % (w/v) D-Glucose
0.15-0.3 % Sodium hydrogencarbonate
Cells were plated onto prepared 35mm polystyrene cell Culture dishes which had been coated with 50jug/ml poly-D-lysine, washed thoroughly and allowed to dry. The cultures were placed in an humidifled atmosphere of 5% CO2 in air at 37°C. Growth medium was replaced after approximately 3 hours.
2.1.2 Maintenance in culture
Medium was replaced approximately 24 hours after plating and thereafter as required; generally every 3-7 days. Where required a cocktail of anti-mitotics was added after a minimum of several days in culture, to give final
concentrations of approximately 0.6/xM each of cytosine-jS-D-arabinofuranoside, 5-fluoro-2’-deoxyuridine and uridine (all supplied by Sigma). Cells were
maintained in culture for 1 - 8 weeks prior to use, though were generally used
after 2-4 weeks in culture.
Cultures are illustrated in figure 2.1. (Age of cultures: 14 DIV)
2.1.3 Acutely dissociated neurones
The above dissociation procedure was also used to obtain neurones for acute use. Cells were plated onto alcian blue-coated borosilicate glass coverslips rather than polylysine-coated plates. Coverslips were dipped into 96% ethanol, flamed and allowed to cool. A few minutes prior to plating, coverslips were covered with 0.1% alcian blue (in dH2 0 ). After 1-2 minutes coverslips were
dipped into distilled water to remove excess alcian blue, and then into growth medium. Cells were plated whilst the coverslips were still wet. Cells were used between 1 and 1 0 hours after plating.
Figure 2.1 Phase contrast micrographs of basal forebrain neurones in dissociated culture. A, Typical culture under low magnification. Several magnocellular neurones are clearly visible (labelled). B, Type II neurone under high magnification. C (overleaf), Type I neurone. D (overleaf), Type IV neurone.
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2.2 Recording setup
2.2.7 General
The microscope was placed on an air table for vibrational isolation and
surrounded by a Faraday cage for electrical isolation. Recordings were obtained using various configurations of the patch-clamp technique (Hamill, Marty, Neher, Sakmann, & Sigworth, 1981).
Current- and voltage-clamps were imposed using a patch-clamp or a switch-clamp amplifier as appropriate. Voltage-clamp was established and
monitored with the aid of two oscilloscopes. Current and voltage commands were generated using either a timer unit or the computer, the latter (connected to the amplifier by the DMA board) using the pClamp suite of software. Amplifier output was lowpass filtered using the amplifier’s integral filter (4- pole Bessel) and an external filter unit (8-pole Bessel) where appropriate. The
signal was recorded using a pen recorder, DAT tape-recorder and/or personal computer. Output to the computer was recorded using pCIamp or axotape software after pre-amplification of the signal where appropriate.
Recordings were obtained from cells only if the seal in the cell-attached configuration was in excess of 1GB and resting membrane potential was at least -60mV. Immediately following recording the electrode was removed from the cell and the junction potential noted. Cells were generally held at -70mV.
2.2.2 Electrodes
Pipettes were fabricated from borosilicate glass capillaries on a vertical electrode puller. For recording electrodes, pipettes were coated in Sylgard silicone elastomer. The Sylgard was cured using a heating coil (constructed in- house). Prior to recording, the electrodes were fire-polished using a
microforge. Electrode, holder and headstage were positioned using a
micromanipulator. Electrode resistance was typically 4-8MB when filled with the intracellular solution given in section 2.2.4 (below).
2.2.3 Bath ground and electrode connections
A bath ground electrode containing 0.9% (w/v) NaCl and 2-3% agarose
contained within a length of borosilicate glass capilary tube connected the bath solution to a 3M KCl solution contained in a separate reservoir. This KCl solution contacted the AgCl terminal of a Ag/AgCl half-cell, which was connected by a length of wire to the earth point of the headstage.
The headstage was in turn connected to the intracellular solution filling the electrode by a length of silver wire with a AgCl-coated surface, thus forming a second Ag/AgCl half-cell. The silver wire was coated with AgCl by electrolysis; the silver wire to be coated was placed in 0.9% (w/v) NaCl and formed the anode. A second silver wire formed the cathode and AgCl coating was acheived by the passage of approximately 8/^A of current for approximately
1 minute. The recording set-up is schematically illustrated in figure 2.2.
2.2.4 Recording solutions Intracellular solution:
Tree calcium concentration 30nM
Approx. 260mOsM
Accompaying ‘modified Krebs’ extracellular solution: K acetate 108mM KCl 15.6mM NaCl 123mM HEPES 40mM KCl 3mM NaOH 10-12mM (pH 7.3) CaCl2 2.5mM EGTA 3mM MgCl2 1.2mM CaCl2 0.822mM* HEPES 5mM (pH7.4) MgCl2 ImM NaHCOg 25mM Mg.ATP 4mM Glucose l l m M Na2.GTP 0. ImM 0.5fiM tetrodotoxin where necessary Approx. 285mOsM
The extracellular solution was continuously gassed with
95% 02/5% C 02.