PROGRAMA UNIVERSIDAD ABIERTA EN LÍNEA

The rCPT was carried out in touchscreen-based operant chambers (Campden Instruments, see chapter 2). Following excitotoxic lesion surgery rats were tested on a range of behavioural manipulations, over a period of around 40 testing sessions. Behavioural manipulations included conditions of reduced SD, congruent and incongruent distraction and high and low event rate (see table 4.2 for experimental outline). Manipulations of SD and distraction were tested under variable and non-variable conditions. Variable conditions involved a range of SDs or distraction types tested randomly and intermixed within a single session, which produces unpredictability. On the other hand,

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non-variable conditions involved a single SD or distraction type tested individually within a single session. During non-variable distraction and high and low event rate manipulations the order of testing was counterbalanced across rats; for example, half of the rats received congruent distraction and the other half incongruent distraction first, to prevent order effects. Note that the novel distraction probe was used in the present experiment, in which flanker distraction stimuli were presented either side of the target, raised by 50% and contrasted to 25% (see chapter 2 and appendix).

4.2.3 Surgery

Rats were divided into four lesion groups: ACC, PL, IL or mixed shams (n=10 in each group).

Statistical analysis confirmed no significant differences between lesion groups on key measures of hit rate, false alarm rate, d’ and C pre-surgery. Rats were anaesthetised with isoflurane gas in oxygen (inducted at 5%, maintained at 2-2.5%) and held in a stereotaxic frame, which was fitted with

atraumatic ear bars and had a digital display console, enabling 10 micron resolution accuracy (David Kopf Instruments; Tujunga, CA, USA). Rats received a pre-surgery analgesic of Metacam (s.c, 1mg/kg, 5mg/ml; Boehringer Ingelheim, Berkshire, UK). A midline incision was made along the scalp to expose the skull, a flat skull measurement was ensured, followed by a craniotomy directly above the infusion sites. Quinolinic acid (0.09M; Sigma Aldrich, UK) dissolved in 0.1M PBS (pH = 7-7.2) was infused bilaterally in the mPFC target sub-region for lesion rats, while for sham rats 0.1M PBS was infused. Table 4.3 displays the stereotaxic coordinates (Paxinos & Watson 1998) and volumes used, which were based on previous studies by Passetti et al. (2002) and Chudasama et al. (2003). Post- infusion the injector was left in place for 2 minutes to ensure infusate dispersion before being slowly retracted. Infusions were made using a 10μl Hamilton precision syringe placed in a Harvard infusion pump (Harvard Apparatus Ltd, Kent, UK), connected to fine bore polythene tubing (0.28mm ID, 0.61mm OD; Portex, Kent, UK) attached to a 31-gauge, stainless steel, bevelled (30⁰) injection needle. Once all infusions were completed the skin was sutured and rats recovered in a heated and ventilated chamber until alert and active. Rats spent one night singly housed and returned to their home cage the next day. Rats were monitored for at least five days post-surgery, in which they received metacam for at least three days (p.o, 1mg/kg, 1.5mg/ml; Boehringer Ingelheim, Berkshire, UK). Seven days post-surgery rats returned to behavioural testing.

mPFC sub-region

Coordinates (mm) Volume of quinolinic

acid (0.1μl/min) ACC AP: +3.2, ML: ±0.7, DV: -1.9 0.3 AP: +2.7, ML: ±0.7, DV: -1.9 0.3 AP: +2.2, ML: ±0.7, DV: -1.9 0.2 PL AP: +3.8, ML: ±0.7, DV: -3 0.4 AP: +2.8, ML: ±0.7, DV: -3.3 0.4 IL AP: +3.0, ML: ±0.7, DV: -4.5 0.4 AP: +2.5, ML: ±0.7, DV: -4.5 0.4

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Table 4.3 Stereotaxic coordinates and volumes of quinolinic acid or sham infused into the ACC, PL or

IL cortex (AP = anteroposterior, ML = medial lateral, DV dorsoventral). AP and ML measurements were taken from bregma on the skull surface, and DV taken from dura, directly above the infusion site.

4.2.4 Drugs

Following testing on behavioural manipulations donepezil, followed by the M4 PAM (VU0467154), was administered under a reduced SD (0.25s). Donepezil hydrochloride (Sigma Aldrich, UK) was dissolved in 0.9% sterile saline and administered in a cross over design at doses of 0 and 1mg/kg, in a volume of 1ml/kg (i.p), 30 minutes prior to testing. VU0467154 (synthesised and provided by Boehringer Ingelheim) was dissolved in 1M hydrochloric acid, 40% (2-hydroxypropyl)-β-cyclodextrin, 1M Sodium hydrochloride and double distilled water. VU0467154 was administered in a cross over design at doses of 0 and 3mg/kg, in a volume of 10ml/kg (p.o: gavage technique), 90 minutes prior to testing. Following each drug day rats were tested with a longer SD (0.5s) and did not receive drug, to ensure a stable performance. A two week washout period occurred following donepezil and before VU0467154 treatment. For dosing protocols for donepezil see chapter 3. The Dosing protocol for VU0467154 were based on the work of Bubser et al (2014) who investigated the pharmacokinetic properties of this new compound, which they have showed to have enhanced in vitro potency and pharmacokinetic properties in rodents, compared to other M4 PAMs.

4.2.5 Statistical analysis

Data were subjected to repeated-measures ANOVA using SPSS version 21 (SPSS Inc, Chicago, IL, USA), with a statistical significance criterion of probability level p<.05. Table 4.2 displays the statistics performed for all behavioural and pharmacological manipulations. Statistical analysis of the three sham groups (ACC sham, PL sham, IL sham) during post-surgery baseline revealed no significant group differences on key measures, and so were combined into one sham lesion group. Lesion ‘group’ always served as a between-subjects factor (4 levels), while the number of ‘days’ the rats were tested for on a particular manipulation always served as a within-subject factor. ‘SD’, ‘distraction’ or ‘event rate’ also served as within-subject factors when appropriate. Significant main effects and interactions were followed up using Sidak’s correction. Due to the different route of administration required for donepezil (i.p) and VU0467154 (p.o), separate vehicle conditions were carried out and therefore data were analysed separately.

4.2.6 Histology

At the conclusion of behavioural testing, animals were administered a lethal dose of sodium

pentobarbitone (Euthatal, 200mg/ml, Merial, UK) and perfused transcardinally with 0.01M PBS (PBS

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two minutes each. Rats were next decapitated, the brain removed and post-fixed overnight in 4% paraformaldehyde, before being cryoprotected in 30% sucrose in 0.01M PBS. Once dehydrated, brains were frozen and sectioned on a cryostat at 60μl thickness. One in every six sections were mounted onto superfrost plus microscope glass slides (Thermo Scientific, UK) and stained with Cresyl Violet for lesion assessment.

1. Rats underwent training on the rCPT (n=40).

2. Rats underwent lesion surgery in which quinolinic acid was infused into the ACC (n=10), PL (n=10) or IL (n=10) cortex, and vehicle into shams (n=10).

3. Rats then underwent a series of behavioural and paramacological manipulations in the following order:

Manipulation Parameters Sample size Repeated measures

ANOVA factors Post-surgery baseline SD = 1s ACC = 10 PL = 10 IL = 10 Sham = 10 w/s: day (4) b/s: group (4) SD Variable SD = 2, 1, 0.5s ACC = 10 PL = 10 IL = 10 Sham = 10 w/s: SD (3) w/s: day (2) b/s: group (4) SD = 4, 1, 0.5s w/s: SD (3) w/s: day (3) b/s: group (4) Non-variable SD = 4, 2, 1, 0.5, 0.25s w/s: SD (5) w/s: day (3) b/s: group (4) Distraction

Variable No, congruent and incongruent

(SD = 4s)

ACC = 10 PL = 9

w/s: distraction (3) w/s: day (3)

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IL = 10 Sham = 10

b/s: group (4)

No, congruent and incongruent (SD = 0.5s)

w/s: distraction (3) w/s: day (3) b/s: group (4)

Non-variable Congruent and incongruent

(SD = 0.5s)

w/s: distraction (2) w/s: day (2) b/s: group (4)

Event rate

Non-variable Low (ITI 5/6s) and high (ITI =

0.5/1s) (SD = 0.5s) ACC = 9 PL = 10 IL = 10 Sham = 9 w/s: event rate (2) w/s: day (3) b/s: group (4) Pharmacology Donepezil (0, 1mg/kg) SD = 0.25s ACC = 10 PL = 10 IL = 10 Sham = 10 w/s: dose (2) b/s: group (4) VU0467154 (0, 3mg/kg) SD = 0.25s w/s: dose (2) b/s: group (4)

Table 4.2 Outline of the behavioural and pharmacological manipulations tested in mPFC lesion

groups on the rCPT. The order of manipulations in the table represents the order in which they were administered. Following surgery recovery, rats were tested on the final stage of rCPT training (‘baseline’: stage 6, SD = 1s). Next, rats were tested under a series of behavioural manipulations including conditions of reduced SD, distraction and event rate. Note, SD and distraction manipulations were tested in variable and non-variable conditions, to assess the effects of unpredictability. All manipulations were carried out for three consecutive days (± one day). Following behavioural manipulations, rats received pharmacology, in which donepezil, followed by the M4 PAM

(VU0467154) were administered under a reduced SD (0.25s). The table also displays the sample size for each behavioural manipulation (any exclusions were due to apparatus related problems). Also displayed are the repeated measures ANOVA factors (w/s within-subjects factor, b/s = between- subjects factor).

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4.3 Results