M . Fendt Brain Research 880 2000 1 –10
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en, Germany. The whole body startle amplitude was 2.5. Drugs
calculated from the difference between the peak-to-peak voltage output of the accelerometer within time-windows
The following drugs were injected into the dPAG of the of 80 ms after and 80 ms before the startle stimulus onset.
rats: 0, 60, and 120 pmol Kainic acid [2S-2a,3b,4b9]-2- The spontaneous motor activity was calculated as the root
Carboxy-4-1-methylethenyl-3-pyrrolidineacetic acid;
mean square value of the accelerometer output, measured AMPA Kainate receptor agonist; Sigma, Deisenhofen,
in a time window of 28 s before the presentation of each of Germany; 0, 50, and 100 nmol NBQX 2,3-Dioxo-6-nitro-
the tone alone startle trials see below. 1,2,3,4 - tetrahydrobenzo[f]quinoxaline - 7 - sulphonamide;
AMPA Kainate receptor antagonist; RBI, distributed by ¨
2.4. Behavioural procedures Biotrend, Koln, Germany; 0, 4.2, and 8.3 nmol Picrotoxin
2
GABA -receptor-Cl -channel blocker; RBI, distributed
A
¨ On the first day, animals were placed into the training
by Biotrend, Koln, Germany; 0, 25, and 50 nmol Piper- boxes and after an acclimatisation time of 5 min, they
idine Piperidine-4-sulphonic acid; GABA -receptor agon-
A 1
¨ received a fear-conditioning training with 10 light
trials. ist; RBI, distributed by Biotrend, Koln, Germany. All
The US was presented during the last 0.5 s of the 3.7 s drugs were dissolved in saline.
light CS at a mean intertrial interval ITI of 2 min range 1.5–2.5 min. On the second day, animals were trained for
2.6. Histology conditioned inhibition using the procedure developed by
2 1
[19]: the rats received 15 noise →
light trials and 5 light
After the tests, the rats were killed by an overdose of trials in a pseudorandomized order mean ITI: 2 min, range
Nembutal. The animals were decapitated, and their brains
2
1.5–2.5 min. A noise →
light trial consisted of a 3.7 s
were removed and immersion-fixed with 8 paraformal- noise following by a 3.7 s light without footshock.
dehyde in PBS with 20 sucrose. Coronal sections of 60 Four hours after this training session, the first test of
m m were taken on a freezing microtome and stained with
fear-potentiated startle and conditioned inhibition occurred. thionin. The injection sites were drawn onto plates from
Two injection cannulae with a diameter of 0.4 mm were the atlas of Paxinos and Watson [39].
inserted into the guide cannulae of the rats. The injection cannulae were connected via a liquid swivel Instech Labs,
2.7. Statistical analysis Plymouth Meeting, PA, USA and by a 50 cm flexible PVC
tubing to two 1 ml syringes Scientific Glass Engineering, Statistical analysis of the data was accomplished by
Weiterstadt, Germany. This method allowed us to inject analysis of variance ANOVA, followed by post hoc
drugs during the test without handling the rats. For testing, Tukey-tests. Trial type and treatment were used as within
the animals were placed into the test cage and after an subjects factors and injection sites were used as between
acclimatisation time of 5 min, 10 initial startle stimuli 10 subjects factors. For all statistical comparisons P,0.05
kHz, 20 ms duration including 0.4 ms rise and fall times, was taken as the criterion for statistical significance. ASR
100 dB SPL, 30 s interstimulus interval were presented to difference scores were calculated for each rat by subtract-
obtain a baseline ASR amplitude. Injections of 0.5 ml drug ing the mean ASR amplitude on tone alone trials from the
solution were given at a rate of 0.1 ml 10 s after the fifth mean ASR amplitude on light tone or noise-light-tone
startle stimulus. The injection cannulae remained in the trials.
brain during the whole test. After the ten initial startle stimuli, each animal received 15 further startle stimuli, one
third presented alone, one third presented 3.2 s after the onset of the light, and one third presented after a light
3. Results
preceded by a 3.7 s noise. All trial types were presented in a pseudo-random order 30 s interstimulus interval with
3.1. Histology the constraint that each trial type occurred only once in
each consecutive three-trial block. Histological analysis revealed that injection sites of 22
The rats received maximal five injections of drug animals were located in the dPAG Fig. 1. Sixteen rats
solutions across five subsequent days. All rats received received injections of Kainic acid into the dPAG, 12 rats
injections of saline and two different drugs in a pseudo- received injections of NBQX, 14 rats received injections of
randomized order into the dPAG. In order to avoid Picrotoxin, and 16 rats received injections of Piperidine.
extinction of fear-conditioning and conditioned inhibition The injection sites of the remaining eight animals were
during the test days, the animals were retrained once daily located in the superior colliculus, in the deep mesence-
4 h before testing. The retraining sessions days 3–6 phalic nucleus, or in the lateral region of the PAG and were
2 1
consisted of 5 noise →
light trials and 5 light
trials in a excluded from further analysis. Since these eight animals
pseudorandomized order with a mean ITI of 2 min range received different drugs, a statistical analysis of their
1.5–2.5 min. behavioural data was not possible.
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Fig. 1. Drawings of frontal sections through the midbrain of the rat depicting the injection sites into the dPAG X, as well as the misplaced injections x. The plates are adapted from Paxinos and Watson [39], the numerals indicate the distance from Bregma. Abbreviations: 3, oculomotor nucleus; BIC,
nucleus of the brachium of the inferior colliculus; dPAG, dorsal PAG; DpMe, deep mesencephalic nucleus; dtg, dorsal tegmental bundle; EW, Edinger–Westphal nucleus; InCo, intercollicular nucleus; MG, medial geniculate nucleus; mlf, medial longitudinal fasciculus; Pn, pontine nuclei; PnO, oral
pontine reticular nucleus; PPTg, pedunculopontine tegmental nucleus; SC, superior colliculus; xscp, decussation of the superior cerebellar peduncle.
3.2. Baseline startle of the previous day were detected ANOVA: F ’s,1, data
not shown. Neither injections of Kainic acid ANOVA: F2,115
0.92, P50.41, NBQX ANOVA: F2,1652.26, P50.14, 3.3. Expression of fear-potentiated startle
Picrotoxin ANOVA: F2,2050.49, P50.62, nor injec- tions of Piperidine ANOVA: F2,1351.00, P50.39 into
Expression of fear-potentiated startle was significantly the dPAG affected the baseline ASR amplitude see black
affected by both, injections of Kainic acid and NBQX into bars in Figs. 2 and 3. No effects of test day or treatment
the dPAG Fig. 2. Whereas Kainic acid dose-dependently
M . Fendt Brain Research 880 2000 1 –10
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Fig. 2. Bar diagrams showing the effects of injections of Kainic acid upper panel and NBQX lower panel into the dPAG on the expression
Fig. 3. Bar diagrams showing the effects of injections of Piperidine of fear-potentiated startle. The bars represent the mean ASR amplitudes
upper panel and Picrotoxin lower panel into the dPAG on expression after tone alone and light-tone trials, as well as the difference scores
of fear-potentiation of the ASR. The bars represent the mean ASR 6S.E.M.. A significant effect of injections on expression of fear-
amplitudes after tone alone and light-tone trials, as well as the difference potentiated startle difference scores is indicated by P,0.01 and
scores 6S.E.M.. P,0.05 repeated-measure ANOVA followed by Tukey-test.
attenuated fear-potentiated startle ANOVA: F2,1153.73, difference scores of noise-light-tone trials ANOVA:
P50.03, NBQX strongly increased fear-potentiated startle F2,1651.68, P50.22, whereas the difference between
ANOVA: F2,1656.83, P50.007. Post-hoc Tukey tests light-tone and noise-light-tone trials was strongly at-
showed a P,0.05 for the comparison between the effects tenuated ANOVA: F2,1655.53, P50.02. But this
of injections of 0 and 120 nmol Kainic acid and a P,0.01 effect was caused by the increase in fear-potentiated startle
for the comparison between the effects of injections of 0 light-tone trials, since the analysis of the percent inhibi-
and 100 nmol NBQX into the dPAG. In contrast, injections tion by the noise CI revealed no significant differences
of both, Picrotoxin and Piperidine Fig. 3, had no effect data not shown; ANOVA: F2,1652.21, P50.14. After
on the expression of fear-potentiated startle ANOVAs: injections of Kainic acid, Picrotoxin, and Piperidine into
F ’s,0.69, P’s.0.52. No effects of test day or treatment the dPAG Fig. 4 and 5, both difference scores were not
of the previous day were detected ANOVA: F ’s,1, data changed ANOVA’s: F ’s,2.40, P’s.0.11. No effects of
not shown. test day or treatment of the previous day were detected
ANOVA: F ’s,1, data not shown. 3.4. Expression of conditioned inhibition of fear-
potentiated startle 3.5. Motor activity
For the analysis of conditioned inhibition, the difference Microinjections of NBQX, Kainic acid, Picrotoxin and
scores of noise-light-tone trials with respect to the tone Piperidine into the dPAG did not affect the motor activity
alone trials as well as the differences between light-tone of the rats in the startle test cage data not shown;
and noise-light-tone trials reflecting the inhibition by the ANOVAs: F ’s,1.64, P.0.24. Explosive motor reactions,
noise CI were compared Figs. 4 and 5. Injections of or defence responses which can be evoked by stimulation
NBQX into the dPAG Fig. 4 had not effect on the of several parts of the PAG using excitatory amino acids or
6 M
Fig. 4. Bar diagrams showing the effects of injections of Kainic acid Fig. 5. Bar diagrams showing the effects of injections of Piperidine
upper panel and NBQX lower panel into the dPAG on conditioned upper panel and Picrotoxin lower panel into the dPAG on conditioned
inhibition of fear-potentiated startle. The bars represent the mean ASR inhibition of fear-potentiated startle. The bars represent the mean ASR
difference scores after light and noise-light trials, as well as their difference scores after light and noise-light trials, as well as their
differences 6S.E.M.. differences 6S.E.M..
GABA receptor antagonist [10,15,41] were not observed in of fear-potentiated startle; likewise, blockade of the
the present study. AMPA Kainate receptors enhanced fear-potentiated startle.
It is important to note that this inhibition of fear-poten- tiated startle by stimulation of the AMPA Kainate re-
4. Discussion ceptors did not reflect the inhibition by an conditioned
