Materials and methods Microiontophoretic application of drugs
268 M
function after brain injury and the adjustments might be buprenorphine Temgesic, 0.05 ml s.c. and allowed to
hastened or improved by appropriate pharmacological recover.
intervention. The reorganisation of the barrel cortex that occurs in
2.1. Unit recording adult rats after chronic vibrissectomy of all but one
whisker, visualised with 2-deoxy glucose [13] was pre- 3–4 days or 20–21 days after lesioning, the rats were
21
vented by locus coeruleus lesions suggesting noradrenergic anaesthetised with urethane 1.0–1.5 g kg
i.p. and again involvement in cortical plasticity [22]. Transhemispheric
placed in a stereotaxic frame. The skull overlying the left cortical reorganisation also depended on central norad-
SmI was removed using a dental drill. The cortex was renergic activity because it was prevented by pretreatment
exposed by a narrow slit in the dura and covered with 4 with DSP4, a neurotoxin destroying the noradrenergic
agar in saline and core temperature maintained at 37618C innervation of the cortex from the locus coeruleus [28].
using a heating lamp. Five ml of 5 glucose solution in A role for acetylcholine ACh in cortical plasticity is
0.18 NaCl was administered s.c. to offset dehydration. also suggested by the observation that the reorganisation in
Single units, isolated by amplitude discrimination, were the somatosensory cortex that would normally occur after
recorded from a site contralateral to the centre of the digit removal or sciatic nerve transection was prevented by
lesion, defined stereotaxically and confirmed electrophy- depleting cortical ACh with an ipsilateral basal forebrain
siologically with cortical evoked potential recording as lesion [12,27]. Sciatic nerve transection also reduced the
described previously [28,29], with the central saline-filled response of somatosensory cortical neurones to ion-
barrel of a 6-barrelled microelectrode impedance 1–3 MV tophoretic application of ACh suggesting the cortical
at 10 KHz, digitised and stored for off-line analysis. reorganisation was associated with changes in cholinocep-
Only spontaneously active neurones which responded to tor sensitivity [17]. The present study examined muscarinic
iontophoretic application of ACh and NA with muscarinic and b-adrenoceptor responses of somatosensory neurones
responses i.e. blocked by atropine and b-adrenoceptor- to iontophoretic application of ACh and noradrenaline
mediated responses i.e. blocked by propranolol, respec- NA 3–4 days and 20–21 days after a unilateral cortical
tively, were analysed see below. The depth of the lesion, i.e. at early and late stages of transhemispheric
electrodes below the pial surface suggested that the cortical reorganisation.
neurones from which recordings were made were mostly located in layers IV–V, and this was confirmed by the
histological data.
2. Materials and methods 2.2. Microiontophoretic application of drugs
Male Sprague–Dawley albino rats B and K Universal, Drugs were administered iontophoretically through the
U.K., weighing 200–250 g, were used. They were housed outer barrels of a 6-barrel glass microelectrode. These
4–6 per cage, with free access to food and water under an contained ACh chloride 0.2 M, pH 4.0, atropine sulphate
alternating 12 h light 0800–2000 and dark cycle in a 0.2 M, pH 4.0, NA hydrochloride 0.1 M, pH 4.0 and
controlled environment 23618C; 45– 60 relative
propranolol hydrochloride 0.1 M, pH 4.0; recordings humidity. The rats were allowed to adapt to their home
were made through the ACSF-filled central barrel. A 10 cage for at least 1 week before being used in any of the
nA backing current was applied to each drug-containing experimental procedures described below.
barrel and compensated through a barrel filled with 2 M Rats were studied either 3–4 days n55 or 20–21 days
NaCl. The agonists, ACh and NA, were each applied for n55 after ablating the hindpaw region in the right SmI,
20 s with ejection currents of 30, 60, 100 and 150 nA. If as previously described [28]; a third group of rats served as
either ACh or NA produced a significant effect on neuro- controls n56. Briefly, the rats were anaesthetised with
nal activity, then the appropriate antagonist was applied, halothane, 1.5–3 in oxygen, mounted in a stereotaxic
by a current equal to the current that produced the response frame, and the skull overlying the right Sm1 was exposed
under investigation, for 25 s commencing 5 s before the under sterile conditions. The hindpaw representation,
agonist. The interval between application of each drug was located from recordings of short latency responses to
a minimum of 3 min and the drugs were applied in the electrical stimulation of the contralateral hindpaw, was
following sequence: ACh alone; Atropine1ACh; NA then ablated to the depth of the white matter using a glass
alone; Propranolol1NA. pipette connected to a vacuum source with pressure
control. After controlling bleeding, the dura was replaced 2.3. Histology
and the scalp incision sutured and dusted with chloro- tetracycline powder Aureomycin, Cyanamid, UK. The
The recording tract was marked at the end of the rats were then given 5 ml i.p. glucose–saline solution 5
experiment by injecting fast green iontophoretically over glucose solution in 0.18 NaCl, and an analgesic,
20 min using 150 nA current while slowly withdrawing the
M . Zarei et al. Brain Research 888 2001 267 –274
269
electrode as described in details previously [28,29]. The centages of neurones responding to different drugs be-
rats were then deeply anaesthetised and perfusion-fixed by tween experimental groups. All variables were tested for a
transcardial infusion of 100 ml of saline followed by 300 two-tail hypothesis.
ml of 4 paraformaldehyde in phosphate buffer 0.1 M, pH 7.4. Sequential vibratome sections 30 mm were cut
and stained with cresyl fast violet and examined with