LICENCIAS CON GOCE DE HABERES II) 3.1 Consideraciones Generales

Operant boxes are commonly used to measure cognitive functions in preclinical rat models as they remove experimenter bias, are capable of recording a range of parameters simultaneously, and may be programmed to be used for a variety of paradigms. A range of operant boxes are commercially available, including the nine-hole box (section 2.7.2; Robbins et al, 1993) where animals may respond to visual or auditory stimuli by nose-poking into an array of holes, and the two-lever „Skinner box‟ chamber (section 2.7.1, e.g. Döbrössy & Dunnett, 1997) where animals respond to visual or auditory stimuli by pressing lateralised levers.

The majority of operant box tasks are designed for either intact rats, or rats with bilateral lesions. However, this thesis project used a unilateral 6-OHDA rat model of PD. To study the effects of acute and chronic dopaminergic medication on motor and non-motor function, it was therefore necessary to identify an operant paradigm which was (i) suitable for rats with unilateral lesions and (ii) able to dissociate between the effects dopamine denervation on motor and non-motor behaviour. One such task is the lateralised choice reaction time (LCRT) task which takes place in nine-hole operant boxes and is described in more detail below.

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1.6.7.1. The lateralised choice reaction time task

The lateralised choice reaction time (LCRT) task (section 2.7.2.2) was originally developed by Carli and colleagues (1985) to distinguish between visual neglect, movement initiation, and movement execution. The task requires rats to respond to illumination of a central hole by making, and maintaining, a centralised nose poke until a visual CS is presented on either the ipsilateral or contralateral side (Carli et al, 1985). In response to the CS, rats must make a lateralised nose poke to gain a pellet reward (Carli et al, 1985). Depending on the version of the LCRT task that is used, this response is either directed to the same hole in which the CS was presented („same‟ version) or the opposite lateralised hole („opposite‟ version; Figure 1.4).

Figure 1.4. A schematic overview of a nine-hole operant box with only three response holes are exposed as is the case in the LCRT task (A), and an example of correct responding in the „same‟ (B) and „opposite‟ (C) versions of the LCRT task. The rats are rewarded for responding to a central light cue by making a sustained nose poke in the central hole until the onset of a lateralised CS, upon which they must either respond in the same response hole the CS was presented in („same‟ version, B) or in the opposite response hole („opposite‟ version, C). Adapted from Dowd & Dunnett, 2005. CS=Conditioned stimulus; LCRT=Lateralised choice reaction time

27 The task provides four main outcome measures: usable trials, reaction time, movement time, and accuracy. „Usable trials‟ refer to the proportion of trials that rats complete by executing a lateralised nose poke in response to the lateralised CS. „Reaction time‟ refers to the time elapsed between the onset of a lateralised CS and discontinuation of the central nose poke. „Movement time‟ refers to the time elapsed between discontinuation of a central nose poke and the completion of a subsequent lateralised nose poke. Finally, „accuracy‟ refers to the proportion of usable trials in which rats direct their lateralised responses into the correct lateral hole following presentation of a lateralised CS.

Using the LCRT task, Carli and colleagues (1985) found that rats that had been pre- trained on the task prior to unilateral 6-OHDA lesions to the striatum exhibited increased reaction time and decreased accuracy when required to respond to contralateral CS by making contralateral nose pokes („same‟ version of the task). No deficit was observed when rats were required to respond to ipsilateral CS by making ipsilateral nose pokes (Carli et al, 1985). A reaction time and accuracy deficit did, however, show when rats were tested on the „opposite‟ version of the task which required them to respond to contralateral CS by making ipsilateral nose pokes and vice versa. In this version of the test, the deficit manifested itself when rats were required to execute contralateral responses to ipsilateral cues. Hence, the deficit was not related to the position of the visual stimulus but rather the direction of the required motor response. Furthermore, when altering the task so that the rats‟ response to a visual cue consisted of making a full turn and pressing the panel on the opposite wall, thus allowing them to respond to contralateral cues by either an ipsilateral or a contralateral turn, the accuracy deficit that had previously been observed following 6-OHDA lesions disappeared (Carli et al, 1985). Together, the data suggested that the deficit was not due to an inability to detect the lateralised CS, but an impaired ability to initiate or execute contralateral movements following 6-OHDA lesions (Carli et al, 1985).

The neurobiology of the deficit was explored in a subsequent study which compared the effects of 6-OHDA lesions to the nucleus accumbens (NAcc) and the striatum on performance on the „same‟ version of the LCRT task (Carli et al, 1989). The previous findings of a 6-OHDA induced deficit in contralateral reaction time and accuracy (Carli et al, 1985) were replicated in rats with intrastriatal lesions but not in the NAcc lesion group (Carli

et al, 1989). This led the authors to hypothesise that dorsal striatal dopamine is essential for

rats‟ ability to initiate contralateral movements (Carli et al, 1989).

Based on the findings of Carli et al (1985, 1989), the LCRT task was later employed by Dowd & Dunnett (2004) as a means of studying the behavioural effects of ventral

28 mesencephalic tissue transplants on rats with unilateral 6-OHDA lesions to the MFB. In line with Carli and colleagues‟ original findings (1985, 1989), dopamine depletion impaired reaction time and accuracy when rats responded to contralateral cues by making contralateral responses („same‟ version). In addition, there was a bilateral increase in movement time that had not previously been reported. The appearance of a movement time deficit may have been due to the greater dopamine depletion induced by MFB lesions, relative to the intra-striatal lesions used by Carli and colleagues (1985, 1989). It may also have been due to the distance between the centre hole and the lateralised hole in which rats‟ responded to a CS, which was greater in Dowd & Dunnett‟s (2004) than in Carli and colleagues‟ (1985, 1989) experiments.

Dowd & Dunnett‟s (2004) publication also differed from Carli and colleagues‟ (1985, 1989) in the manner in which they illustrated the post-lesion behaviour. Whereas the publications by Carli and colleagues (1985, 1989) presented bar graphs showing the post- lesion performance averaged across a block of testing, Dowd & Dunnett (2004) used line graphs to show the post-lesion behaviour as a function of time. When presenting the contralateral accuracy using a line graph it became apparent that the contralateral accuracy deficit manifested itself gradually (Dowd & Dunnett, 2004). Specifically, the lesion group was able to accurately respond to contralateral stimuli on the first day of post-lesion testing but thereafter displayed a gradual decrease in contralateral accuracy over the subsequent four days of testing. Interestingly, following a break from testing and reintroduction to the task eight weeks post-lesion, the accuracy of contralateral responding on the first day of testing returned to the initial post-lesion levels. Similar to what had previously been observed a gradual decline in contralateral performance was then observed over the following four days of testing. This phenomenon could not be explained by a continued loss of striatal dopamine since the lesion was complete at the time of testing. Furthermore, if the deficit was purely motor mediated the impairment would be expected to have been stable from the outset of post-lesion testing. Hence, the accuracy deficit could not be explained by Carli and colleagues‟ (1985, 1989) original hypothesis of an impaired ability to initiate contralateral movements (Carli et al, 1989; Brown & Robbins, 1989)

The finding of a gradual decline in performance rather than a stable impairment was replicated in a subsequent experiment which compared the effects of unilateral 6-OHDA intra-striatal and MFB lesions on performance on the „same‟ version of the LCRT task (Dowd & Dunnett, 2005a). Both lesions caused a gradual impairment in contralateral reaction time, movement time, and accuracy when tested two weeks post-lesion, although the accuracy deficit was more pronounced in the MFB lesion group. When re-tested four months

29 post-lesion the intra-striatal lesion group seized showing a deficit in their reaction time, movement time, and accuracy while the MFB lesion group continued being impaired on all parameters. Interestingly, in line with previous findings, the contralateral accuracy of the MFB lesion returned to initial post-lesion testing levels on the first day of testing after which a second gradual decline in performance was observed (Dowd & Dunnett, 2005a). A follow- up experiment, including rats with MFB or intra-striatal lesions, was conducted in which the „opposite‟ version of the LCRT task was used, i.e. where rats were required to respond to ipsilateral CS by making a contralateral response and vice versa. In this version of the task, both the MFB and intra-striatal lesion groups exhibited impaired accuracy when required to respond to ipsilateral stimuli by making a contralateral response (Dowd & Dunnett, 2005b). In contrast to the earlier findings, which were obtained using the standard „same‟ version of the task (Dowd & Dunnett, 2005a), this accuracy deficit remained stable in both lesion groups when re-tested four months post-lesion (Dowd & Dunnett, 2005b).

From a methodological perspective, the described studies suggest that the standard „same‟ version of the LCRT task is appropriate for long-term experiments including rats with lesions to the MFB, but not to the striatum The difference between the two lesions‟ effect on LCRT task performance may be due to the extent of dopamine denervation they induce in the SNc or VTA, which is greater following a MFB than intra-striatal lesion, or the ability of the MFB lesions to also affect prefrontal dopamine levels (Dowd & Dunnett, 2005a).

From a theoretical perspective, the most notable difference between Carli and colleagues‟ (19895, 1989) and Dowd & Dunnett‟s (2004, 2005a,b) publications was the demonstration of a gradually emerging contralateral accuracy deficit in the latter publications. In Dowd & Dunnett‟s (2004, 2005a,b) publications, this deficit disappeared on the first day of re-testing rats following a three months test-free interval. Dowd & Dunnett (2007) noted that this re-emergence of contralateral responding resembled „spontaneous recovery‟ of contralateral accuracy. „Spontaneous recovery‟ is one of the hallmarks of extinction. It refers to the tendency of a conditioned response (CR) to reappear in animals that have undergone extinction when these animals are reintroduced to the same task following a period of not being tested. In addition to „spontaneous recover‟, extinction is also associated with „reinstatement‟ and „renewal‟. „Reinstatement‟ refers to the re-appearance of a CR in animals exposed to the US between extinction and retention testing. „Renewal‟ refers to the tendency of a CR to reappear in animals that have undergone extinction when retested in a different environment (Myers & Davis, 2002). Based on the gradual decline in contralateral accuracy, which was similar to what occurred when physically removing the sucrose pellet reward

30 following a correct contralateral response (Dowd & Dunnett, 2007), and the „spontaneous recovery‟ like re-emergence of responding that was observed when lesion rats were retested after a test-free interval, Dowd and Dunnett (2007) hypothesised that the contralateral accuracy deficit observed in lesion rats represented extinction. This was hypothesised to be driven by an ablation of the dopaminergic reward signal that otherwise occur following presentation of a US (Schultz, 2010) in the lesion rats.

The LRCT task data reviewed above demonstrate the presence of motor deficits in the form of increased reaction and movement times following unilateral 6-OHDA lesions, as well as non-motor deficits that may reflect extinction (Carli et al, 1985, 1989; Dowd & Dunnett, 2004, 2007). The LCRT task may thus be used to measure deficits to both motor and non- motor function. This made it an appropriate task when conducting experiments designed to dissociate between the effect of dopaminergic drugs on motor and non-motor behaviour in rats with unilateral 6-OHDA lesions as part of this thesis project.