Materials and methods ments of the rats resulted in changes of the voltage output

2 M first hints to brain structures possibly involved in con- available. The experiments were done in accordance with ditioned inhibition of fear-potentiated startle: 1 Fendt ethical guidelines for the care and use of experimental [22] showed that a blockade of GABA receptors within animals and were approved by the local council of animal A ¨ ¨ the dorsal region of the periaqueductal gray dPAG care Regierungsprasidium Tubingen, ZP 4 96. attenuates expression of conditioned inhibition of fear- potentiated startle; 2 Heldt and Falls [29] demonstrated 2.2. Surgery that electrolytic lesions of the brachium of the colliculus inferior block the expression of conditioned inhibition of The animals were anaesthetised with ketamine xylazine fear-potentiated startle. 9:1, 100 mg kg, i.p.. Two stainless guide cannulae Whereas the brachium of the colliculus inferior is a part diameter: 0.7 mm were implanted bilaterally into the of the auditory pathway mediating the noise CI [37,50], the brain using following stereotaxic coordinates of Paxinos dPAG is a brain structure which is not directly involved in and Watson [39]: rostrocaudal: 26.3 mm, mediolateral: the processing of the auditory CI, but is well known for 6 0.6 mm, dorsoventral: 25.0 mm relative to Bregma. modulation of fear and anxiety [5,24,48]. Beside its The guide cannulae were fixed to the skull with dental possible role in conditioned inhibition of fear-potentiated cement and three anchoring screws. After the surgery and startle [22], the dPAG plays a role in the expression of between the experiments, stainless steel stylets diameter: fear-potentiated startle. Walker and colleagues [49] demon- 0.4 mm were inserted into the guide cannulae to maintain strated that dPAG stimulation by injections of Kainic acid patency. All subjects were allowed 4 days recovery before block fear-potentiated startle whereas dPAG lesions pre- training and testing. vent the loss of fear-potentiated startle seen after training with high intensity USs [11]. Furthermore, we previously 2.3. Apparatus demonstrated [25] a projection from the dPAG to the caudal pontine reticular nucleus, a crucial part of the The rats were trained in two identical, acoustically 3 primary startle circuit [32]. A number of other studies isolated dark boxes 38360328 cm . The floor of the using different animal models to investigate fear-related boxes was composed of stainless steel bars spaced |15 behaviours showed that the dPAG is strongly involved in mm apart, through which the unconditioned stimulus US, the expression and modulation of fear [20,28,30,31,34– a 0.5 s, 0.6 mA footshock could be administered. The 36,44]. It is important to note that with the methods used footshocks were produced by a shock generator custom- ¨ in the studies cited above it is not possible to distinguish made at the University of Tubingen located outside the between the dorsomedial and the dorsolateral part of the chamber. The conditioned stimulus CS for fear-con- PAG, two areas which can be clearly differentiated [2]. ditioning paired with the US was a 3.7 s white light 1 The aim of the present study was to investigate the role produced by a 15 W bulb light trials, the conditioned of AMPA Kainate and GABA receptors within the dPAG inhibitor CI, predicting that the light is not followed by a A in the expression and conditioned inhibition of fear-poten- footshock, was a 3.7 s, 70 dB white noise produced by a 2 tiated startle. First, we tested whether the blockade of loudspeaker noise → light trials. The bulbs and the fear-potentiated startle which were reported after injections loudspeakers were located on the top of the boxes. The of the AMPA Kainate receptor agonist Kainic acid into the presentation of the US, CS and CI were controlled by a dPAG reflects conditioned inhibition. In addition, the microcomputer and an appropriate interface Hortmann effects of injections of the AMPA Kainate receptor an- universal function synthesiser, Hortmann, Neckartenzling- tagonist NBQX were investigated. Second, we attempted en, Germany. to replicate the blockade of conditioned inhibition of fear- To measure fear-potentiated startle and conditioned potentiated startle reported after GABA receptor blockade inhibition, the animals were tested in two identical test A by Picrotoxin within the dPAG and to confirm these results chambers. The rats were placed in wire mesh cages 203 3 by an opposite effect after GABA receptor stimulation by 10312 cm with a steel floor, which were put up on a A Piperidine. piezoelectric accelerometer custom-made at the University ¨ of Tubingen. The accelerometer was located inside a 3 sound-attenuated test chamber 100380360 cm . Move-

2. Materials and methods ments of the rats resulted in changes of the voltage output

of the accelerometers. These signals were amplified, 2.1. Animals digitised and fed into a computer for further analysis. A loudspeaker, set up at a distance of 40 cm from the wire A total of 30 male Sprague Dawley rats supplied by mesh cage, delivered the acoustic startle stimuli and a Charles River, Sulzfeld, Germany, weighing between 250 continuous white background noise 55 dB SPL, RMS. and 350 g were used. They were housed in groups of 5–6 The presentation of the acoustic stimuli was controlled by animals under a continuous light–dark cycle lights on a microcomputer and an appropriate interface Hortmann from 7:00 A.M. to 7:00 P.M.. Food and water were freely universal function synthesiser, Hortmann, Neckartenzling- M . Fendt Brain Research 880 2000 1 –10 3 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