neurotoxicity and mechanisms of induced hyperexcitability

tral regions of the mPFC (the prelimbic and the infralimbiccortices) have been associated with diverse emotionaland cognitive processes [43, 46, 47]. The ventral mPFCis also of interest, as it has been strongly implicated inthe expression of behavioural and autonomic responsesto emotionally relevant stimuli [48]. As dysfunction ofthe prefrontal cortex has been repeatedly implicated inthe pathophysiology of schizophrenia [49-52], studies inthe rat have focused on elucidating the role of this regionin paradigms such as locomotor activity and prepulse inhibition.Dopaminergic lesions and intra-mPFC infusionof selective dopamine receptor antagonists have beenreported to disrupt prepulse inhibition [53, 54], whereasthe intra-mPFC infusion of amphetamine has been shownto decrease systemic amphetamine-induced increases inlocomotor activity in the open field test [55]. However,none of these studies have addressed the importance ofserotonergic innervation of the mPFC in neural circuitryinvolved in the regulation of motor behaviour and prepulseinhibition. Therefore, the present study investigatedthe effects of local lesions of serotonergic projections intothe mPFC on psychotomimetic drug-induced locomotorhyperactivity and prepulse inhibition.MATERIALS AND METHODSExperimental animalsA total of 25 male Sprague-Dawley rats (Departmentof Pathology, University of Melbourne), weighing 250-300 g at the time of surgery, were used in this study. Theanimals were housed under standard conditions in groupsof two or three with free access to food and water. Theywere maintained on a 12 h light/dark cycle (lights on at0700 h) at a constant temperature of 21ºC. One week priorto the surgical procedure, the animals were handledeach day over a five-day period. The experimental protocoland surgical procedures were approved by the AnimalExperimentation Ethics Committee of the University ofMelbourne, Australia.Drugs and solutionsD-amphetamine sulphate (Sigma Chemical Co., St.Louis, MO, USA) and phencyclidine HCl (PCP, Sigma)were dissolved in a 0.9% saline solution and injected subcutaneously(s.c.) into the nape of the neck. DesipramineHCl (Sigma) was dissolved in distilled water and injectedintraperitoneally (i.p) 30 min prior to the neurotoxin microinjection.All doses are expressed as the weight of thesalt and were administered in an injection volume of 1 ml/kg body weight. The serotonergic neurotoxin, 5,7-dihydroxytryptamine(5,7-DHT) (Sigma), was dissolved in 0.1%ascorbic acid (BDH Chemicals, Kilsyth, VIC, Australia) insaline to prevent oxidation of the neurotoxin. Carprofen(50 mg/ml, Heriot AgVet, Rowille, VIC, Australia) was dilutedin 0.9% saline to a dose of 5 mg/kg and injected s.c.immediately after the surgical procedure.Surgical procedureThe rats were pretreated with 20 mg/kg desipramine,30 min prior to surgery, to prevent the destruction of noradrenergicneurons by 5,7-DHT [56]. The rats were subsequentlyanaesthetised with sodium pentobarbitone (60mg/kg i.p., Rhone Merieux, QLD, Australia). The rats weremounted in a Kopf stereotaxic frame (David Kopf Instruments,Tujunga, CA, USA) with the incisor bar set at –3.3mm [57]. The skull surface was exposed, and a small holewas drilled. A 25 gauge stainless-steel cannula, which wasattached to a 10 μl glass syringe and connected via polyethylenetubing mounted in an infusion pump (UltraMicro-Pump, World Precision Instruments, Sarasota, FL, USA),was lowered into the mPFC. With bregma set to zero andthe stereotaxic arm at 0º, the coordinates were as follows:mPFC lesions (n=13 for behavioural experiments, n=2 forhistology): 3.2 mm anterior, 0.7 mm lateral and 4.5 mmventral to bregma. A volume of 0.5 μl of 5,7-DHT (5 μg/μl)was infused over a period of 2 min on each side. Sham-operatedcontrols (n=10) underwent the same surgical procedureand received an equal volume of vehicle solution.The injection volumes and rate of infusion were selectedto minimise non-specific damage at the site of injection.Movement of the meniscus in the cannula was monitoredto ensure successful infusion. After infusion, the cannulawas left in place for a further 2 min to avoid backflow ofthe solution up the injection path. After lesioning, theskin was closed with silk-2 sutures (Cynamid, BaulkhamHills, NSW, Australia), and the animals were administered5 mg/kg of carprofen, a non-steroidal, anti-inflammatoryanalgesic, to reduce post-operative inflammation and discomfort.The rats were placed on a heated pad until theyrecovered from the anaesthesia. After the surgery, the ratswere allowed to recover for two weeks, during which theywere handled regularly and health checks were made twoto three times a week.Experimental design and apparatusBehavioural tests were performed starting two weeksafter the surgery, and each session included random numbersof 5,7-DHT-lesioned rats and sham-operated rats.Locomotor activity was monitored using eight automatedphotocell cages (31 x 43 x 43 cm, h x w x 1, ENV-520, MEDAssociates, St. Albans, VT, USA). The position of the rat atany time was detected with sixteen evenly spaced infraredsources and sensors on each of the four sides of the monitor.The addition of a photobeam array above the subjectadded a second plane of detection to the system to detectrearing and vertical counts. This infrared beam array thusdefined an X, Y and Z coordinate map for the system. Thesensors detected the presence or absence of the infraredbeam at these coordinates. Every 50 msec, the softwarechecked for the presence or absence of the infrared beamat each sensor, allowing for the very precise tracking ofthe movement of a subject. Several types of behaviouralresponses were recorded, including distance moved, ambulation,stereotypy and rearing. Ambulatory counts con-13