neurotoxicity and mechanisms of induced hyperexcitability

sisted of consecutive interruptions of at least four beamswithin a period of 500 ms. Small, repetitive beam breakswithin a virtual box of 4 x 4 beams around the rat wererecorded as stereotypic counts. Recordings of photocellbeam interruptions by the rat were taken every 5 minand stored by the computer software. Three locomotoractivity tests were done after treatment with saline, 0.5mg/kg of amphetamine or 2.5 mg/kg of phencyclidine administeredin a random order. These locomotor activitytests were done with three to four day intervals to preventhabituation due to repeated testing and to allow forclearance of the drugs. Prior to any drug manipulation,the rats were placed in the locomotor photocell cages for30 min to establish baseline locomotor activity and allowfor habituation to the test environment. After 30 min ofspontaneous baseline activity, the rats were injected andlocomotor activity was recorded over a further 90 min,generating a total session time of two hours. For the purposeof this paper, locomotor activity data were expressedas cumulative data from 30 min periods and presented asa time course of distance moved during the 30 min beforeinjection and 90 min after injection.After locomotor activity experiments, rats were testedfor prepulse inhibition. This was done using six automatedstartle chambers (SR-LAB, San Diego Instruments, SanDiego, CA, USA) consisting of clear Plexiglas cylinders, 9cm in diameter, resting on a platform inside a ventilated,sound-attenuated and illuminated chamber. A speakermounted 24 cm above the cylinder produced both continuousbackground noise at 70 dB and the various acousticstimuli. Whole-body startle responses of the animal in responseto acoustic stimuli caused vibrations of the Plexiglascylinder, which were then converted into quantitativeresponses by a piezoelectric accelerometer unit attachedbeneath the platform. The percent prepulse inhibition wascalculated as 100 x {[pulse-alone trials – (prepulse + pulsetrials )] /(pulse-alone trials)} [31]. At least one day beforethe prepulse inhibition testing, rats underwent a pretestsession where they were exposed to the testing cylindersand the testing protocol for the first time. This session wasconducted to allow rats to habituate to the testing environment.Each rat was placed into the chamber for a 5 minacclimation period with a 70 dB background noise levelthat continued throughout the session. A single prepulseinhibition session lasted for about 45 min and consistedof high- and low-intensity stimulus combinations with acontinuous background noise of 70 dB. The session startedand ended with a block of ten pulse-alone trials of 115dB. These blocks, together with twenty pseudo-randomlypresented pulse-alone trials during the prepulse inhibitionprotocol, were used to calculate the basal startle reactivityand startle habituation. Prepulses were presented for 20 msand differed in intensity. Prepulse inhibition was assessedby the random presentation of 115 dB pulses, ten each ofprepulse-2, -4, -8, 12 and –16 and ten ‘no-stimulus’ trials.For example, prepulse-8 (PP8) is a 20-ms prepulse of 8 dBabove the background noise, i.e., 78 dB, followed 100 mslater by a 40-ms 115 dB pulse [58]. The interval betweentrials varied (10 - 37 s) to prevent conditioning of the responses.A microcomputer and an interface assembly thatcontrolled the delivery of acoustic stimuli digitised and recordedthe readings.Tissue preparation for histology and high pressureliquid chromatography (HPLC)At the end of the experiments, rats were killed by decapitationand the brains were removed from the skull.The brains were placed on a cold plate. First, the frontalcortex was dissected bilaterally [59], and then the mPFCwas dissected out. For the histological assessment of thelocation of the injection sites, 20-μm-thick sections of themedial prefrontal cortex of two mPFC-lesioned rats werecut on a cryostat and mounted onto gelatin-coated glassslides. The sections were then stained with cresyl violet(ProSciTech, Thuringowa, QLD, Australia) and examinedmicroscopically to verify the location of the tips of theinfusion cannulas.The HPLC measurements of the tissue serotonin (5-HT)concentration were carried out in 23 animals. The dissectedstructures were weighed and stored in Eppendorf tubes at–80°C until the biochemical assays were performed. The tissuesamples were homogenised in 500 μl of 0.1 M perchloricacid by ultrasonication and centrifuged at 15,500 g for 5min. A 50 μl aliquot of the supernatant was injected into ahigh pressure liquid chromatography (HPLC) system to determinethe content of 5-HT (ng/mg tissue of wet weight).The HPLC system consisted of a Waters Model 510 Solventdelivery system, a Waters U6K injector, an Alphabond C18125A 10 U 150* 3.9 mm column and a Column & Spectra-Physics 970 D-A1 fluorescence spectrometer. The outputsignal from the fluorescence detector was analysed with thechromatography software package, 810 Baseline, version3.31. The mobile phase used consisted of 9.8 g/l KH 2PO 4, 1.0g/l Na 2EDTA, 5% acetonitrile and 1 ml/l triethylamine. ThepH of the mobile phase solution was adjusted to 3.0 with 1M HCl. Subsequently, the solution was filtered and degassedand delivered to the HPLC at a flow rate of 1 ml/min. Priorto sample testing, the following standards for 5-HT were runthrough the system: 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml and 200 ng/ml. Calibration curves were constructed, andthe level of 5-HT in tissue samples was calculated relativeto these standards. Each run lasted 8 min and the retentiontime for serotonin was 2.6 min.Statistical analysisData were expressed as the mean ± the standard errorof the mean (SEM). All of the statistical analyses were performedusing the statistical software package SYSTAT 9.0(SPSS Inc., Chicago, IL, USA). All of the data were analysedusing an analysis of variance (ANOVA) with repeatedmeasures where appropriate. In the locomotor activity experiments,data were summed in 30-min blocks, and theseblocks were used to assess the main effects of the lesiontype (group), the treatment with amphetamine or phency-14