Variable measurements

From the number of hits and misses during target presentation a hit rate was generated; a higher hit rate indicating good performance (see table 2.2). From the number of false alarms and correct rejections during non-target presentation a false alarm rate was generated; a lower false alarm rate indicating good performance. Hit rate and false alarm rate alone do not provide a full representation of performance. For example, a high hit rate in combination with a high false alarm rate is not

necessarily good performance and instead may indicate a general increase in responding which is non-selective to the target stimulus; while a lower hit rate in combination with a lower false alarm rate is not necessarily bad performance, and may indicate a general decrease in responding which is non- selective to the target stimulus. To overcome this, two further variable measures were generated which take into account both hit rate and false alarm rate scores by applying signal detection theory (Frey & Colliver 1973). One of these, d’, provides a measure of discrimination sensitivity by assessing the ability of rats to visually discriminate between the target and non-target; a higher value indicating good discrimination. The other is C, which provides a measure of response bias by assessing the willingness of an animal to make a response in general (at both the target and non-target); a higher value indicating more liberal responding, a lower value indicating more conservative responding.

Other measures recorded for analysis were the number of premature/perseverative responses during the ISI. Due to the continuous nature of the task the premature and perseverative aspects are unable to be pulled apart; therefore this measure was not split by SD/distraction condition for analysis. Measures of response speed were assessed by mean hit response latencies following hits and false alarm response latencies. A gross measure of motivation was provided by mean reward retrieval latencies.

Table 2.2 Key variable and other measures used for statistical analysis on the rCPT.

Key variable measures Other measures

𝐻𝑖𝑡 𝑟𝑎𝑡𝑒 = 𝐻𝑖𝑡𝑠

𝐻𝑖𝑡𝑠 + 𝑀𝑖𝑠𝑠𝑒𝑠

Premature/perseverative responses

𝐹𝑎𝑙𝑠𝑒 𝑎𝑙𝑎𝑟𝑚 𝑟𝑎𝑡𝑒 = 𝑀𝑖𝑠𝑡𝑎𝑘𝑒𝑠

𝑀𝑖𝑠𝑡𝑎𝑘𝑒𝑠 + 𝐶𝑜𝑟𝑟𝑒𝑐𝑡 𝑟𝑒𝑗𝑒𝑐𝑡𝑖𝑜𝑛𝑠

Hit response latency

𝑑′_{= 𝑧(𝐻𝑖𝑡 𝑟𝑎𝑡𝑒) − 𝑧(𝐹𝑎𝑙𝑠𝑒 𝑎𝑙𝑎𝑟𝑚 𝑟𝑎𝑡𝑒) False alarm response latency}

𝐶 =𝑧(𝐻𝑖𝑡 𝑟𝑎𝑡𝑒) + 𝑧(𝐹𝑎𝑙𝑠𝑒 𝑎𝑙𝑎𝑟𝑚 𝑟𝑎𝑡𝑒)

2

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2.3 5-Choice Serial Reaction Time Task (5-CSRTT)

2.3.1 Behavioural apparatus

Experiments using the 5-CSRTT were carried out in both touchscreen and non-touchscreen, five-hole operant chambers (figure 2.4), both described previously (Horner et al. 2013; Bari et al. 2008). The touchscreen-based operant chambers were the same Campden Instruments chambers as the ones described for the rCPT. A black Perspex mask was positioned in front of the screen with five holes cut out creating distinct response windows. Each window was 2.5cm², 0.8cm away from the screen and 1.5cm above the grid floor. The illumination of a response window in white represented the visual stimulus. The five-hole operant chambers, of which there were 12, were Med Associates (Georgia, VT, USA), controlled by whisker software (Cambridge Cognition, UK; Cardinal & Aitken 2010). The chambers (height: 28cm, width: 25cm, depth: 28cm) were made of aluminium with a clear Perspex side wall and door. The front wall was curved in a concave manner in which 5 response apertures were located. Each aperture was 2.5cm², 2cm deep and 2cm above the grid floor. At the rear of the apertures were yellow LEDs which acted as the visual stimuli; at the aperture entrance, positioned horizontally, were infrared photocell beams which recorded entries. The rear wall featured the food magazine, in which 45mg food pellets were delivered (Sandown Scientific, Middlesex, UK). Infrared photocell beams located horizontally on the magazine entrance recorded entries to and exits from the magazine. The chambers also contained a house light and barred grid floor and were enclosed within fan-ventilated, light and sound attenuating boxes.

Figure 2.4 Schematic diagram of the (a) five-hole and (b) touchscreen-based 5-CSRTT chambers

(images taken from Bari et al, (2008) and provided by Campden Instruments, respectively).

2.3.2 Behavioural training

Rats were trained over a series of 12 stages to sustain visual spatial, divided attention on a horizontal array of five apertures and detect and report the pseudo-random presentation of a brief visual

stimulus (figure 2.5 and table 2.3). The program was implemented with the house light on in the five-

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hole and off in the touchscreen-based chambers (to be consistent with the battery of touchscreen- based paradigms). The training protocol has been described previously (Carli et al. 1983; Bari et al. 2008). In brief, rats were initially habituated to the chambers for one 20-minute session, in which chambers were powered on, with no programme running, and ten food pellets available in the magazine. In the five-hole chambers, two pellets were also available in each aperture; this was not feasible in the touchscreen-based chambers. Following habituation rats began training. Sessions began with the illumination of the house light (five-hole chambers only) and food magazine and the delivery of a food pellet. The entry of the rat’s nose to the magazine to collect the pellet terminated the magazine light and initiated the first trial, which began with a fixed ITI. Rats were required to detect the pseudo-random presentation of a visual stimulus in one of five spatial apertures and respond in the corresponding aperture within a fixed LH period, which begins at stimulus onset, to earn a food pellet (‘correct response’). Food pellets were delivered in conjunction with the illumination of the magazine light. Trials were continuous, and so following a correct response the entry of the rat’s nose to the magazine to collect a pellet terminated the magazine light and initiated the next trial. A

response made during the ITI (‘premature response’), in a non-corresponding aperture (‘incorrect response’) or no response (‘omission’) resulted in a 5 second timeout (TO) period in which the house light was terminated (five-hole) or switched on (touchscreen-based) and no food pellet delivered, to discourage inappropriate responding. Following the TO period, the magazine illuminated for rats to nose-poke which initiated the next trial. Each training session consisted of a maximum of 100 trials, with each trial representing an opportunity to earn a food pellet, within a maximum of 30 minutes. Premature responses were deemed an incomplete trial and did not count towards the 100 trials. Perseverative responses were responses made in an aperture following a correct or incorrect response and were recorded but not punished. Over the 12 training stages the SD, LH and ITI reduced. In the final stage of training, stage 12 (SD: 0.5s, LH: 5s, ITI: 5s), a criterion of ≥70 percent accuracy and ≤20 percent omissions was required. The accuracy criterion is slightly lower than that reported in Bari et al. and was used due to this being the performance level most rats could acquire to a stable level. Most rats completed training within 50-60 sessions. Towards the end of training rats often completed sessions in ~20 minutes. When stage 12 was achieved rats were rested and given refresher sessions twice weekly until the entire cohort completed training. Once all rats had acquired stage 12 they were tested for at least three consecutive days to ensure a stable baseline performance before any behavioural, pharmacological or surgical manipulations.

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Figure 2.5 Flowchart of the rodent touchscreen-based/five-hole 5-CSRTT. The first trial is initiated via

a nose-poke to the magazine and begins with a fixed ITI (5s). Rats were required to sustain visual spatial, divided attention on a horizontal array of five spatial apertures and detect and report the presence of a brief visual stimulus presented pseudo-randomly. A response in the corresponding aperture within the LH period resulted in a food pellet delivery (‘correct response’). A response made prior to stimulus onset (‘premature response’), in a non-corresponding response aperture (‘incorrect response’) or no response (‘omission’) resulted in a TO period in which the house light was

terminated (five-hole) or illuminated (touchscreen-based) for 5s and a food pellet was not obtained. Reward collection (on correct trials) initiated a new trial, while a nose-poke to the illuminated magazine initiated a new trial after a TO. Sessions were 30 minutes in duration, with a maximum of 100 trials.

Stimulus (0.5s) and LH (5s) onset Fixed ITI (5s)

Magazine entry initiates new trial, location pseudo-randomly selected Start ‘Correct’ Food pellet delivered Collect pellet Time out (5s) ‘Incorrect’ ‘Omission’ ‘Premature’

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Stage SD(s) LH(s) ITI(s) Criterion

1 30 30 2 ≥ 30 correct 2 20 20 2 ≥ 30 correct 3 10 10 5 ≥ 50 correct 4 5 5 5 ≥ 50 correct 5 2.5 5 5 ≥ 70 percent accuracy ≥ 50 correct 6 1.25 5 5 ≥ 70 percent accuracy ≥ 20 percent omissions ≥ 50 correct 7 1 5 5 ≥ 70 percent accuracy ≥ 20 percent omissions ≥ 50 correct 8 0.9 5 5 ≥ 70 percent accuracy ≥ 20 percent omissions ≥ 50 correct 9 0.8 5 5 ≥ 70 percent accuracy ≥ 20 percent omissions ≥ 50 correct 10 0.7 5 5 ≥ 70 percent accuracy ≥ 20 percent omissions ≥ 50 correct 11 0.6 5 5 ≥ 70 percent accuracy ≥ 20 percent omissions ≥ 50 correct 12 0.5 5 5 ≥ 70 percent accuracy ≥ 20 percent omissions

Table 2.3 5-CSRTT 12 stage training protocol (adapted from Bari et al. 2008). Rats were trained over

12 stages to detect and report the presence of a brief visual signal in one of five apertures. The stimulus duration (SD), limited hold (LH) and inter-trial-interval (ITI) reduced over stages.

2.3.3 Probes

Manipulations were often implemented under challenging conditions of reduced SD. The reduced SD probe involved sessions of 0.5s (stage 12 ‘baseline’ SD) intermixed with reduced SDs of 0.25 and 0.125s.

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2.3.4 Variable measurements

The number of correct, incorrect, omission and premature responses were used to generate four variable percent measures (table 2.4). Percent accuracy is the key attentional sensitivity measure which assesses signal detection; percent correct and omissions can also to some extent be considered measures of attentional performance. On the other hand, percent premature responses provides a measure of impulsivity. Percent accuracy measures the number of correct responses divided by correct and incorrect while percent correct also takes omissions into account. Percent omissions measures the number of omissions divided by correct, incorrect and omissions. Percent premature responses measures the number of premature responses divided by all other responses; as percent premature responses occur prior to stimulus onset they were not split by SD for analysis. The number of additional responses made in response apertures following a correct or incorrect response were recorded as perseverative responses, which are often interpreted as a measure of compulsivity. The mean correct and incorrect response latencies, as well mean reward retrieval latency were also recorded.

Table 2.4 Key variable and other measures used for statistical analysis on the 5-CSRTT.