Behavioural training

Rats were trained over a series of 6 stages to sustain visual focussed attention on a single response window and detect, discriminate and report the brief presentation of an infrequent patterned target stimulus, presented in sequence with a range of non-target stimuli (figure 2.2 and table 2.1). Note that the rCPT was implemented with the house light off, which is consistent with the battery of other touchscreen-based paradigms. The training protocol has been described previously (Mar et al. 2017). 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. Following habituation rats began training on stage one; rats were trained to focus on a single response window outlined in white (7.5 x 7.5cm), positioned central on the screen, 3.5cm above the floor grid. The response window was present on screen for the entire duration of sessions throughout training. Trials began with a varied inter-stimulus interval (ISI = 2/3s) which remained constant throughout training. Rats were required to detect and report the presentation of a white square stimulus (stimulus duration

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(SD) = 10s) within a limited hold period (LH = 10.5s), which began at stimulus onset, to receive a food pellet (‘hit’). A hit made before the end of the SD resulted in the immediate removal of the stimulus from the response window, while a hit made after the SD was up, but within the LH, was carried out when the stimulus was no longer on screen. Following a hit, a food pellet was delivered in conjunction with a 1s tone and the illumination of the magazine. Trials were continuous in nature and only paused following a ‘hit’, in which a nose-poke to the magazine to collect the food pellet initiated the next trial. The entry of the rat’s nose to the magazine to collect the food pellet terminated the magazine light and initiated a 2s after reward pause for pellet consumption, before initiating the next trial. If a response was not made during the LH (‘miss’) a new trial began automatically. Any responses at the blank response window during the ISI re-set the ISI timer, delaying the next stimulus onset, to

discourage inappropriate responding at the response window. The criterion for stage one was 100 hits within a 45 minute session. The session terminated either when 100 hits were achieved or after 45 minutes. In stage two, the white square stimulus was replaced with the rat’s designated target stimulus (vertical or horizontal patterned stimulus, counterbalanced across rats) and presented for a shorter SD of 5s with a 5.5s LH; the same criterion as stage one applied.

In stage three, rats were trained to detect, discriminate and report target stimulus presentation in sequence with a novel non-target stimulus (snowflake; 50/50 probability); presented for a shorter SD of 3s with a 3.5s LH. Rats were required to respond at the target stimulus within the LH to receive a food pellet (‘hit’) and to withhold responding during non-target stimulus presentation (‘correct rejection’). If a response was made at the non-target stimulus during the LH (‘false alarm’), the stimulus was immediately removed from the response window (if the SD timer was not up) and a correction trial loop was initiated in which a series of non-target stimuli were presented until a correct rejection was made. The correct trial loop delayed subsequent target stimulus presentation, to discourage inappropriate responding. From stage three onwards trials were uncapped and rats could earn up to 150 pellets within a 45 minute session -- it was not possible to earn this amount of pellets before 45 minutes was up -- meaning all rats tested for the full session and could not finish early. The criterion for stages three to six were a hit rate of ≥0.5 and d’ ≥1 (see 2.2.4 variable measurements). In stage four, four novel non-target patterned stimuli replaced the snowflake stimulus and were

presented randomly and in sequence with the target stimulus (30% probability); stimuli were

presented for 2s with a 2.5s LH and the same criterion as stage three applied. In stages five and six the SD and LH were reduced to 1.5s (SD) with a 2s LH and 1s (SD) with a 1.5s LH, respectively. When stage six criterion was achieved, rats were rested and given refresher sessions twice weekly until the entire cohort completed training. Most rats completed training within ~21 sessions. Once all rats had acquired the task they were tested for at least 3 consecutive sessions to ensure a stable baseline performance before any behavioural, pharmacological or surgical manipulations.

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Figure 2.2 Flowchart of the rodent continuous performance task (rCPT). Trials began with a varied

inter-stimulus interval (ISI). Rats were required to sustain visual, focussed attention on a single response window and report the brief presentation of an infrequent patterned target stimulus

(horizontal/vertical patterned stimulus, counterbalanced), presented randomly and in sequence with a range of non-target stimuli (30% probability). During target stimulus presentation a response (‘hit’) or no response (‘miss’) occurred, the former resulting in a food pellet. During non-target stimulus presentation a response (‘false alarm’) or no response (‘correction rejection’) occurred. A false alarm resulted in a correction trial loop in which a string of non-target stimuli were presented until a correct rejection was achieved, delaying target stimulus presentation. A response to the response window during the ISI period (‘premature/perseverative response’) reset the ISI timer, to discourage

inappropriate responding. Trials were continuous in nature and only paused following a ‘hit’, in which

Start

Stimulus (SD = 1s) and limited hold onset (LH = 1.5s) Varied ISI (2/3s)

New trial, stimulus selected randomly

Target

Response:

‘hit’

Food pellet delivered

Collect pellet Correction trial: Non-

target stimulus selected randomly Response: ‘false alarm’ ‘Premature/ perseverative responses’

Stimulus offset and LH continues

No response:

‘miss’ No response: _rejection’ ‘correct

Non-target Target / Non-target stimuli

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a nose-poke to the magazine to collect the food pellet initiated the next trial. Sessions were 45 minutes in duration, with no trial cap.

Stage Stimuli SD (s) LH (s) ISI (s) Target

probability Max no. food pellets Criterion (days to criterion) 1 10 10.5 2/3 n/a 100 Hits = 100 (~3) 2 5 5.5 2/3 n/a 100 Hits = 100 (~1) 3 3 3.5 2/3 50% 150 Hit rate ≥ 0.5 d’ ≥ 1 (~4) 4 2 2.5 2/3 30% 150 Hit rate ≥ 0.5 d’ ≥ 1 (~6) 5 1.5 2 2/3 30% 150 Hit rate: >0.5 d’ >1 (~4) 6 1 1.5 2/3 30% 150 Hit rate ≥ 0.5 d’ ≥ 1 (~3)

Table 2.1 rCPT six stage training protocol. Rats initially learned to detect and then discriminate a

target stimulus presented randomly and in sequence with 4 other non-target stimuli. Stimulus duration (SD) and limited hold (LH) reduced over training sessions whilst the varied inter-stimulus interval (ISI) remained the same. Rats acquired the task in ~21 sessions. In this example the horizontal patterned stimulus is the target stimulus.

2.2.3 Probes

Successful attentional function requires the ability to detect information rapidly, as well as inhibiting potentially distracting information. Therefore, manipulations were often implemented under

Target Target

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challenging conditions of reduced SD and/or distraction; of which have been commonly reported to increase attentional load and tax attentional function in humans. The rCPT reduced SD probe was often variable and involved sessions of 1s (stage 6 ‘baseline’ SD) intermixed with reduced SDs of 0.6 and 0.2s.

The rCPT distractor probe (figure 2.3) is a variant of the Eriksen Flanker task (Eriksen & Eriksen 1974). Sessions involved the presentation of response-congruent and response-incongruent flanker distractors. Distractors were positioned on the left and right of the central response window and were the same size (7.5 x 7.5cm). The response windows for the distractors remained on screen

throughout the entire session. Responses at the distractors were recorded but had no consequences; they were present only to guide performance. During distractor trials the stimulus duration was fixed. The distractors were initially positioned directly either side of the central response window and in matching contrast to the central response window stimuli. This was later altered so that they were positioned either side of the central response window, raised by half of the height of the stimulus (50%) and contrasted to 25%. This was found to tax attention (reduce discrimination of the target and non-target: d’), whilst also reducing direct contact with the distractors in the form of distractor

responses (see appendix 1). Congruent-distractor trials involved the presentation of the same target or non-target stimuli as that presented in the central response window; this trial type could be easier as rats receive three times the signal of ‘target’ or ‘non-target’. Incongruent-distractor trials involved the presentation of non-target stimuli distractors on target trials (which may impair performance in the form of reduced hit rate and d’) and target stimuli distractors on non-target trials (which may impair performance in the form of increased false alarm rate and reduced d’). Within a session, congruent- and Incongruent-distractor trials were often intermixed with no-distractor trials, in which the distractor response windows remained present but with no stimuli presented. Note that correction trials following a false alarm, were always no-distractor trials.

No-distractor Congruent-distractor Incongruent-distractor

ISI

Target trial

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Figure 2.3 Diagram of the distractor trial types. Two response windows were positioned either side of

the central response window and raised by half of the height of the stimulus (50%). All response windows remained on screen throughout the entire session. Congruent- and incongruent-distractor trial types were often intermixed with no-distractor trials. Distractors were contrasted to 25% and responses at them had no consequences.