C .M. Thiel et al. Brain Research 882 2000 112 –119
113
havioural habituation during re-exposure to the same open The rats were anaesthetised with a combination of Ketavet
field on the following day. Smaller cholinergic increases 0.9 ml kg and Rompun 0.4 ml kg, and were implanted
were, however, also found in a control group, which was with guide canulas, which consisted of a 15 mm thin wall
handled but not exposed to the open field, indicating that stainless steel canula 22 G with a thread in the top [7,41].
aversive aspects of handling might have contributed to The canula was aimed above the ventral hippocampus A:
increased extracellular ACh levels in this task [47]. 2
6.0, L: 64.8, V: 23.2 mm and was secured to the skull Therefore, the present study was intended to explore in
with dental cement. After surgery, the animals were more detail hippocampal cholinergic activity in relation to
handled daily and allowed to recover for at least 4 days aversive stimulation using a conditioning paradigm. A first
before the beginning of microdialysis. aim was to investigate the behavioural and neurochemical
effects of aversive stimulation a footshock, which was 2.1.2. Dialysis procedure
paired with a neutral stimulus a tone. Rats were exposed On the day preceding the experiment, a microdialysis
to a single tone shock pairing and a tone alone 2 h later, probe of concentric design length of membrane: 4 mm
while extracellular hippocampal ACh levels and behav- was inserted into the guide canula. The probes were
ioural activity were measured. A control group was only self-manufactured as described previously [41]. After
exposed to the tone. Behavioural testing in rats often insertion the probe was connected to a microinfusion pump
involves a constant masking background noise during the via a liquid swivel mounted on a balanced arm above the
1
experiment. Therefore, a second aim was to investigate cage. The probe was perfused with Ringer solution Na
1 11
2
whether such background noise might influence behav- 147 mmol, K
4 mmol, Ca 2.25 mmol, Cl
155 mmol ioural and neurochemical responses to aversive stimulation
at room temperature. In order to obtain detectable levels of in the above conditioning procedure. Thus, a third group of
ACh, 0.3 mM neostigmine was added to the Ringer rats was exposed to a single tone shock pairing and 2 h
solution. After probe insertion, the animal was placed later to a tone alone while being constantly exposed to
individually into the experimental cage 26328328 cm background noise.
which was situated in a sound attenuated chamber. Two The results showed that background noise prevented the
speakers were mounted on top of the cage for delivery of increase in hippocampal ACh levels to the tone shock
auditory stimuli. The animal was kept under a 12 h stimulation without, however, preventing the uncon-
light dark cycle luminous density: 60 Lux and had free ditioned behavioural response. We therefore performed a
access to food and water. second, behavioural experiment to investigate in more
On the next day, dialysis was performed between 9.30– detail the consequences of enduring background noise.
14.00 h. Samples were taken manually every 10 min. After Assuming that the septohippocampal cholinergic system is
collection of three baseline samples the animals were involved in learning, we asked whether the observed lack
exposed to the following procedures: of ACh activation in noise exposed animals would also
result in reduced learning. To keep conditions similar to 2.2. Experimental manipulations
the above in vivo microdialysis study, we chose shuttle box avoidance learning, a task, where rats are trained to
escape avoid an aversive footshock, which is signalled by •
Tone shock without background noise n57. Rats a tone. Half of the rats were exposed to constant back-
were exposed to a single tone shock pairing tone: ground noise during avoidance learning, while a control
1600 Hz for 3 s, 115 dB, followed immediately by a group was exposed to the learning paradigm without
60 s 0.3 mA scrambled footshock; constant current background noise. We hypothesised, that an attenuated
shock generator, 521 C, Campden Instruments; pa- cholinergic hippocampal reactivity in noise exposed ani-
rameters determined by piloting according to an mals would result in impaired active avoidance learning.
aversive conditioning procedure. Two hours later, the tone was presented again without shock. This 2 h time
window was chosen since hippocampal ACh levels
2. Materials and methods were expected to be at baseline 2 h after the tone
shock administration. 2.1. Experiment 1: neurochemical and behavioural
• Tone control group without background noise n55.
effects of aversive stimulation and the effects of Animals were exposed to a 3 s 115 dB tone, which
background noise was repeated after 2 h.
• Tone shock with background noise n510. In this
2.1.1. Subjects and surgery group, animals were treated like those in group A, i.e.
Male Wistar rats 240–370 g obtained from the Tier- exposed to a single aversive tone shock pairing and a
¨ versuchsanlage of the University of Dusseldorf were used.
tone alone after 2 h. Additionally, they were also They were housed individually under standard laboratory
exposed to a constant broadband background noise 70 conditions and were allowed free access to food and water.
dB, main frequencies 200–6.300 Hz which started 2 h
114 C
before beginning of dialysis i.e. 7.30 am and lasted analysed by means of independent t-tests. A P-value of
throughout the whole testing session. 0.05 was required for significance.
2.3. Experiment 2: effects of background noise on active 2.2.1. Behavioural data analysis
avoidance learning Behaviour was videotaped and analysed subsequently
with the help of a semi-automated computer system. The following behavioural measures were analysed at stimulus
2.3.1. Subjects relevant time points in blocks of 10 min corresponding to
Twenty male Wistar rats weighing between 270 and 330 the dialysis samples. Locomotion was scored as the
g at the beginning of the experiment were used. They were number of crossings of virtual lines dividing the cage into
housed individually under standard laboratory conditions four quadrants. Rearing was scored as the number of times
and allowed free access to food and water. All animals the rat reared up on its hindlimbs, irrespective of whether
were handled daily for 3 days prior to behavioural testing. the animal showed on- or off-wall rearing. Since the most
obvious response to our shock was twitching, this be- 2.3.2. Apparatus
haviour was scored as the time which the animal spent The shuttle box was constructed of grey plexiglas and
twitching or shaking the fore- and hindlegs in response to measured 66 cm333 cm wide339 cm high. The floor was
the footshock. made of 2 mm diameter stainless steel rods spaced 1.5 cm
apart. The box was divided into two equal compartments 2.2.2. Neurochemical data analysis
by a 5 cm high plexiglas barrier. Each compartment could Microdialysis samples were assayed for ACh levels
be electrified separately through a shock scrambler 521 using HPLC–ED [10,41]. The chromatographic separation
C, Campden Instruments. A speaker was mounted in the of ACh was achieved by a 80 mm long column filled with
centre on the top of the box for delivery of auditory Chromospher 5C
and loaded with sodium-dodecylsul- stimuli.
18
fate. An enzyme reactor was attached to this column, which was filled with LiChrosorb-NH
activated by
2
2.3.3. Procedure glutardialdehyde, and which contained 80 units of acetyl-
Rats were tested under two different conditions accord- cholinesterase and 40 units of choline oxidase co-valently
ing to the microdialysis experiments: One group of ani- bonded to the stationary phase. Passing the enzyme
mals, termed ‘noise’ n510, was exposed to a constant reactor, ACh was converted to hydrogen peroxide, which
broadband background noise 70 dB which started 2 h was detected at a VT-03 electrochemical flowcell potential
before beginning of behavioural testing and which lasted set at 500 mV; ANTEC Decade detector. The mobile
throughout the testing session. The other group of rats, phase was composed of 0.1 M K HPO
and 1 mM
2 4
termed ‘no noise’ was not exposed to background noise tetramethyl-ammonium-chloride adjusted to pH 8.0 with
n510. KH PO and delivered at a flow-rate of 0.5 ml min. The
2 4
Rats were placed in the shuttle box and allowed to freely detection limit for ACh was approximately 15 fmol in-
explore the apparatus for 180 s. Then, they received 20 jection. The neurochemical data, which were not corrected
shuttle trials, where they were trained to terminate a shock for dialysis recovery, are presented and analysed in terms
by jumping over a barrier to the adjoining compartment. of percentages of baseline.
Each trial began with a 3 s 115 dB tone followed by a 0.3 mA scrambled footshock according to the microdialysis
2.2.3. Histological analysis experiment. If the animal crossed the barrier during the
At the end of the experiment, the animals were deeply tone, the stimulus was terminated and no shock was
anaesthetised with Nembutal and perfused transcardially delivered avoidance response. If the animal crossed the
with saline followed by 10 phosphate buffered formalin. barrier during shock delivery, an escape response was
The brains were removed, sliced on a cryotome and stained measured. If the rat failed to cross, the shock was
with cresyl violet for analysis of probe placement. Only terminated after 20 s escape failure. After 30 s, the next
animals with correct probe placements located within the trial was initiated.
ventral hippocampus were used for further data analysis [38,47].
2.3.4. Data analysis Behaviour was videotaped and analysed subsequently.
2.2.4. Statistical analysis Escape latencies were pooled in blocks of 5 trials. If a rat
Neurochemical data were analysed by means of ANOVA failed to escape during the 20 s of the shock application, a
for repeated measures with Greenhouse Geisser correction latency of 23 s 3 s tone120 s shock was scored. Rats that
using time as the repeated measure 23 levels. Reported failed to show the appropriate behavioural responses i.e.
values refer to the main effect of experimental manipula- sitting on the barrier or jumping out of the apparatus were
tion. Behavioural data during tone shock application were excluded from analysis.
C .M. Thiel et al. Brain Research 882 2000 112 –119
115
2.3.5. Statistical analysis Escape behaviour was analysed for both groups using
ANOVA for repeated measures with the blocks of trials 4 levels as factor. Post hoc analysis of significant effects
was performed using Tukey tests. A P-value of 0.05 was required for significance.
3. Results
