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
3. Results
reference to an atlas of rat cerebral cortex [30]. Responses of 61 neurones from control rats, 60 neurones
2.4. Statistical analysis from rats 3–4 days post lesion and 56 neurones from rats
20–21 days post lesion to iontophoretic application of NA Responses of single neurones to iontophoretic applica-
and ACh each 30–150 nA were studied. Background tion of drugs were determined from analysing the averages
firing rates of the neurones in rats 3–4 days post lesion and of 3 histograms for each trial, each consisting of 3
rats 20–21 days post lesion 3.061.2 and 2.961.4 spikes
21
consecutive 20 s blocks before, during and after drug s
6S.D., respectively did not differ from those in the
21
administration. The mean firing rates before 20 s, back- age-matched control rats 3.161.2 spikes s
6S.D.. ground and during iontophoretic drug application 20 s
There were no differences P,0.05 in the mean latencies were noted and compared. Averages of 3 drug administra-
and durations of neuronal responses to ACh and NA in the tions were used for statistical analysis of drug effects,
different groups Table 1. significance of effects being calculated using Wilcoxon
tests for significant changes in neuronal activity P,0.05 3.1. Effects of ACh
between the background activity and the activity during the period of chemical stimulation. Latency and duration of
About two-thirds 61–69, depending on the ejection significant changes in neuronal firing after the beginning of
current of all somatosensory neurones recorded, respond- the stimulation was automatically detected and noted
ed to iontophoretic application of ACh with an atropine- Spike Neurosoft, Elprog Ltd., Moscow. The ratios of
sensitive response in control rats see Fig. 1 for an example excitatory to inhibitory responses E I ratio were also
of PSTH. The responses were largely excitatory and calculated. Firing rates are given as means6S.D. spikes
dose-dependent i.e. the E I ratio increased from 1.1:1 at 30
21
s unless otherwise stated.
nA to 8.6:1 at 150 nA Figs. 2 and 3a. This was mainly To compare firing rates, latencies to onset of drug action
due to neurones, which were inhibited by ACh at the lower and durations of drug action between control and ex-
currents being excited at higher currents, because the total perimental groups an independent ANOVA were used. A
number of responsive neurones was relatively unaffected
2
x cross-tabulation test was used to compare the per-
Fig. 4. About 10 of neurones showed a mixed bimod-
Table 1 Latencies and, in parentheses, durations of neuronal responses to iontophoretic application of ACh or NA at each ejecting current n5number of neurones.
There are no significant differences between control group and the experimental groups Ejection
Mean response latencies and durations6S.D. current
s nA
Control 3–4 post lesion
20–21 days post lesion n561
n560 n556
ACh 30
7.564.3 5.365.9
6.065.1 8.865.6
10.465.1 7.065.3
60 5.164.0
7.266.9 5.364.8
11.968.7 11.7610.7
10.668.7 100
5.764.0 4.964.5
6.064.7 15.569.2
15.9611.7 12.168.1
150 5.164.3
5.264.8 4.264.9
19.365.0 14.368.0
14.2610.6 NA
30 4.762.6
6.464.7 6.464.6
8.264.8 9.764.6
9.065.2 60
6.964.8 9.666.2
7.165.1 10.263.5
7.663.2 9.065.4
100 6.164.3
5.164.6 5.764.8
12.766.0 12.067.5
7.263.9 150
6.064.1 5.565.1
7.065.7 11.563.6
9.364.6 10.666.5
270 M
Fig. 1. A typical neurone in layer IV of SmI showing an excitatory response to iontophoretic application of ACh. The response is blocked by
iontophoretic application of atropine commencing 5 s prior to application of ACh and ceasing at the same time as the application of ACh.
al response but this percentage dropped to 2 at the highest current Fig. 4.
Three to 4 days after cortical ablation, the percentage of neurones responding to ACh decreased to between 14 and
22 of the proportion of spontaneously active neurones, depending on the current P,0.05. The increase in the
E I ratio described in control rats was not seen, there was
Fig. 2. Ratios of excitatory to inhibitory responses to iontophoretic
no dose–response effect and the proportions of excitatory
application of ACh a and NA b in hindpaw region of somatosensory
and inhibitory reactions were similar at most currents
cortex of control rats either 3–4 days or 20–21 days after ablation of the
except 60 nA Figs. 3a and 4. Fewer neurones responded
corresponding region of the contralateral cortex; negative values represent
in a bimodal manner to ACh than in control rats, about
inhibitory to excitatory ratios. ACh application usually excited neurones
10 cf. 2 or less P,0.02.
in control rats but in the rats 3–4 days post lesion, excitatory and inhibitory responses occurred in approximately equal proportions. In the
The total proportion of neurones responding to ACh had
rats 20–21 days post lesion, excitatory responses to ACh were only
returned to the control level by 3 weeks after the lesion
dominant at the lowest ejection current. NA usually evoked an inhibitory
and a limited dose–response effect was observed; 46 of
response in control rats but an excitatory response in the rats 20–21 days
neurones responded at 30 nA and 60 responded at 60 nA,
post lesion. Intermediate effects were seen in the rats 3–4 days post
but further increase of current did not increase the propor-
lesion. No. of neurones561 in control rats, 60 in rats 3–4 days post lesion and 56 in rats 20–21 days post lesion.
tion of responsive neurones. However, increasing current had little effect on the proportions of neurones excited by
ACh but increased the proportion of inhibitory responses Fig. 4 i.e. E I ratio dropped considerably P,0.05;
from 3.7:1 at 30 nA to 0.7:1 at 100 nA Fig. 3a. The total
0.5:1 at higher currents and this was entirely due to an population of neurones exhibiting bimodal responses was
increase in the proportion of neurones excited by NA similar to that in control rats.
because the proportion which were inhibited was unaffect- ed Fig. 3b. Depending on the ejection current, 4 to 9
of neurones showed a bimodal response. 3.2. Effects of NA
Three to 4 days after lesion, the proportion of cortical neurones which were sensitive to iontophoretic application
In control rats, between 48 and 62 of neurones, of NA decreased significantly P,0.05, to between 14
depending on the current applied, exhibited a propranolol- and 24 depending on the ejection current, and the dose–
sensitive response to NA. The responses were predomi- response effect was abolished Fig. 5. The lowest dose of
nantly inhibitory, particularly at the lowest current when NA, which evoked the greatest proportion of inhibitory
17 out of 22 NA-sensitive neurones 77, responded with responses in control rats, evoked the greatest proportion of
inhibition and only 1 neurone was excited Fig. 5. The excitatory responses in rats 3–4 days post lesion, so that
E I ratio increased from about 0.05:1 at 30 nA to about the E I ratio increased P,0.001 from 0.05:1 in control
M . Zarei et al. Brain Research 888 2001 267 –274
271
Fig. 5. Proportion of neurones in hindpaw region of SI responding to iontophoretic application of NA at ejection currents of 30, 60, 100 and
Fig. 3. A typical does dependent response of a neurone in layer IV of rat 150 nA. The proportion of responding neurone at each ejection current
SmI. decreased markedly 3–4 days after lesioning the contralateral cortical
area but had recovered 3 weeks later. In reverse to changes in the effect of ACh in rats 20–21 days post lesion, NA which were usually inhibitory
in control rats, showed a high proportion of excitatory responses to NA; indicating that unlike ACh, NA effect may reverse during cortical
plasticity after the contralateral lesion. No. of neurones561 in control rats, 60 in rats 3–4 days post lesion and 56 in rats 20–21 days post lesion.
rats to 4.3:1 in the rats 3–4 days post lesion Fig. 3b. The proportion of excitatory responses decreased at higher
ejection currents. The proportion of neurones with bimodal responses was much lower than that in control rats.
Three weeks after the lesion, the percentage of NA- sensitive neurones had returned to the control level e.g.
52 at 30 nA compared with 48 in control rats at the same ejection current Fig. 5. Increasing the ejection
current to 60 nA, significantly increased the proportion of responsive neurones, evidence for the return of a dose–
response effect. However, further increase of the current did not significantly alter the proportion of responding
neurones Fig. 5. Similarly, the E I ratio of neuronal responses increased P,0.05 from 1.9:1 at 30 nA to 3.9:1
at 60 nA but decreased to 1.0:1 after the current increased to 150 nA Fig. 3b. This was due to an initial increase in
Fig. 4. The proportion of neurones in hindpaw region of control rats and
the proportion of neurones, which were excited by NA at
rats 3–4 days and 20–21 days after lesioning of the corresponding
the lower ejection current without affecting the proportion
contralateral cortex, responding with an inhibitory, excitatory or mixed biphasic response to iontophoretic administration of ACh at 30, 60, 100,
of neurones, which were inhibited; however the latter
150 nA for 20 s. The total proportion of neurones responding to
increased at the higher ejection currents with a concomi-
acetylcholine was reduced 3 days after the lesioning. Although this had
tant decrease in the former Fig. 5. The majority of
recovered by 3 weeks, there was a shift from predominantly excitatory
neurones showed an excitatory response to NA at each of
responses in controls to a greater proportion of inhibitory responses. No.
the different ejection currents. It is noteworthy that neuro-
of neurones561 in control rats, 60 in rats 3–4 days post lesion and 56 in rats 20–21 days post lesion.
nal responses to NA changed from inhibitory to excitatory
272 M
reported previously [16]. The predominantly excitatory response is consistent with a previous study, which de-
scribed their occurrence in all cortical layers, especially in laminae Vb and VIb [16], although the proportion of
excitatory responses was higher in the present study. A modulatory role of ACh in SmI plasticity is suggested by
the finding that it enhanced the strength of the neuronal responses to peripheral stimulation and glutamate for up to
1 h after iontophoretic application [20]. It has been proposed that the modulatory response to ACh enables
cortical neurones to rapidly alter the magnitude of their responses to afferent stimulation [15]. These rapid changes
in receptive field size may then be stabilised by longer lasting changes because ACh is also involved in the
development of new receptive fields [27,12].
The involvement of the cholinergic system in cortical plasticity of adult rat somatosensory cortex was suggested
by changes in cholinergic markers choline acetyltrans- ferase, acetylcholine esterase, high-affinity choline uptake
in the hindlimb receptive fields of the rat SmI, 1 to 63 days after unilateral transection of sciatic nerve [25]. These
studies showed that 1 day after sciatic nerve transection there was a 30 reduction in high-affinity choline uptake
in layer V of the contralateral somatosensory cortex and, at 3 days, a 15 reduction in ChAT activity. In rat SmI,
unilateral sciatic nerve transection altered binding of the
3
M1-selective ligand, [ H]pirenzepine, in bilateral hindlimb region of somatosensory cortex, indicating the involvement
of muscarinic M1 receptors in somatosensory cortical plasticity [10]. Other studies suggested that the changes in
Fig. 6. An example of neuronal response to application of NA. Most
cholinergic markers in the rat barrel cortex after vibrissec-
neurones in SmI showed an inhibitory response to iontophoretic applica-
tomy were related to altered morphology and not to
tion of NA in control rats. However during transhemispheric cortical reorganization, excitatory neuronal responses increased markedly.
abnormal functioning of the barrel cortex as a result of the reduced sensory input from the vibrissae [8].
Iontophoretic application of NA onto somatosensory over a period of 3 weeks after the lesion see Fig. 6 for an
neurones in urethane-anaesthetised rats has previously example.
been shown to produce inhibitory and, occasionally, excitatory responses [1]. The nature of the neuronal
responses, their latencies and the bell-shaped pattern of
4. Discussion PSTH constructed during application of NA, were con-
