POSIBILIDAD DE REFORMAR EL MARCO NORMATIVO INTERNACIONAL

The first study in this experimental chapter attempted to replicate previous observations that STN/ZI-lesions impair the formation of attentional set. After two sessions of testing on the 7-stage task, lesioned rats did not demonstrate a cost of shifting attentional set. Unfortunately, sham-operated control rats also failed to demonstrate a convincing ID- ED difference and there was no statistical support for group differences in set formation. The most parsimonious conclusion for the first study of this experiment would be that control rats did not present sufficiently robust evidence of set formation and that whilst STN lesions may have disrupted set formation, there is no convincing evidence of absence, owing to a small sample size.

Despite the mixed evidence from the first study, it has been shown that control rats that have failed to demonstrate an ID-ED difference in the 7-stage task benefit from multiple ID stages in a subsequent task – facilitating a significant cost of set-shifting (Lindgren et al., 2013). Therefore, the second study of this experimental chapter presented multiple ID stages, whilst investigating the mechanisms of set-formation using the 11-stage task with the same cohort of rats. The result of the ID5-ED comparison in Study II was similar to the ID-ED comparison from Study I: the study failed to demonstrate robust and statistically-supported evidence of a cost of set-shifting in controls and compromised performance in lesioned rats. Overall, performance for lesioned rats did not differ from control rats, which included performance on the multiple ID stages, reversal stages, the probe, and the bi-conditional discrimination. The lack of clear behavioural effects is most likely due to a lack of consistent lesion damage, in both size/extent and symmetry, which ultimately led to a loss of statistical power owing to a reduced group size (n=5).

3.4.1 Limitations and considerations

The histology for the ‘lesioned’ group indicated marked asymmetry. Lesions were only discernible at all in five rats, of which only one had clear bilateral damage; the remaining four rats had only unilateral discernible lesions. Another large concern pertained to the consistency in achieving damage to cells in the STN; over half of the rats that received ibotenic acid infusions (n=6) expressed ‘indeterminate’ lesion damage. These rats

102

were not ‘intact’, in that they had calcium deposits in the MGP (which we have previously reported to accompany subthalamic cell loss; Phillips and Brown, 1999; Xia, Dhawan, Tait & Brown, unpublished), along with postoperative chewing. Nevertheless, there was no evidence of cell loss in the STN or ZI of these rats. It is highly likely that the failure to detect a robust behavioural deficit in the lesion group is predominantly due to a small sample size, but also due to the nature of the lesion damage being largely unilateral, and therefore permitting sufficient intact tissue to support function.

3.4.1.1 Unilateral vs bilateral inactivation

Considerable research from obtained from studies with Parkinson’s patients has detailed a significant difference between unilateral and bilateral approaches to HFS for treatment of motor symptoms. For example, bilateral HFS provides greater improvements in motor functioning than unilateral stimulation (Bastian, Kelly, Revilla, Perlmutter, & Mink, 2003; Goelz et al., 2016; Kumar et al., 1999), but induces cognitive decline not found in unilateral stimulation (Amara et al., 2012), including deterioration in non-verbal recall performance (Williams et al., 2011) along with impaired verbal fluency and trouble inhibiting dominant verbal responses on the Stroop test (York et al., 2008). Furthermore, bilateral HFS – and not unilateral HFS – in Parkinson’s patients, impairs cognition on the “n-back task” (see Owen et al., 2005), which tests working memory, encoding and updating (Alberts et al., 2008). Cognitive performance for unilaterally-stimulated PD patients did not differ from patients without stimulation (Alberts et al., 2008), highlighting the importance of bilateral STN inactivation in generating cognitive impairment. This consideration may have contributed to the result found in this chapter; given that of the majority of the lesioned rats presented with unilateral damage (4 of the 5 rats), it is possible that sufficient damage was not incurred in order to evidence a cognitive deficit.

In their study, Alberts et al. (2008) postulated that the increased activation in the dorsolateral prefrontal cortex (dlPFC) observed during performance of working memory tasks, such as the n-back task (see Jansma, Ramsey, Coppola, & Kahn, 2000; Owen, McMillan, Laird, & Bullmore, 2005) might be disrupted during STN-HFS, owing to the established fronto-subthalamo projections (see: Nambu, Tokuno, & Takada, 2002). It is possible that disruption of information processing and response selection in parts of the

103

STN which contribute to cognitive impairments perhaps by incorrectly shaping output activity (Alberts et al., 2008). This consideration is also consistent with research in rats, in which the observed cognitive deficits produced by bilateral lesions to the STN could largely stem from the disruption of the STN–medial prefrontal cortex (mPFC) circuit (Chudasama, Baunez, & Robbins, 2003). Disconnection lesions of the mPFC-STN (i.e., lesioning the STN and mPFC unilaterally, but in opposite hemispheres) considerably impaired rats’ discriminative accuracy and also increased perseverative responding and response latencies in the 5CSRTT; moreover this functional deficit was remarkably similar to the impairment seen after bilateral STN lesions (Chudasama et al., 2003). Conversely, unilateral STN lesions induced only a mild deficit, slightly increasing both premature and perseverative responses compared to sham-operated animals, without affecting discriminative accuracy and response latency. Chudasama et al. (2003) thus concluded that the cognitive deficits – particularly those of attention and executive function – exhibited by bilateral STN lesioning depend upon the cortico-subthalamic projection, and additionally their research implicitly illustrates the importance of achieving bilateral STN lesions when examining the role of the STN in cognition. The fact that accuracy and latency were unaffected following unilateral lesions also weakens that position that unilateral lesions may induce an attentional impairment, which further suggests that perhaps bilateral lesions are mandatory to obtain cognitive deficits.

Cognitive research, in which lesions to the rat STN are made, predominantly employ bilateral lesions to produce behavioural deficits (See Baunez & Lardeux, 2011; Jahanshahi et al., 2014). Unilateral lesioning has been used in animal studies to dissociate generalised motor effects of striatal dopamine depletion (e.g., hyperkinesia) from response- specific initiation effects, by comparing ipsilateral vs contralateral motor performance biases (Phillips & Brown, 1999; see section 1.8.1), whilst consequently serving as a means to evaluate the quantitative differences between unilateral and bilateral lesioning. Rats with unilateral lesions of the STN in Phillips & Brown (1999) did not exhibit impairments in accuracy, omissions, or perseverative responding in nose-poke visuo-spatial discrimination tasks, as typically expected of rats with bilateral lesions to the STN in similar tasks (Baunez

104

(anticipatory) responses. The present evidence regarding the effects of bilateral lesions on latency performance in visuo-spatial reaction time tasks is mixed, with evidence that lesioning both ‘speeds up’ (Baunez et al., 1995), and ‘slows down’ (Baunez & Robbins,

1997; 1999) reaction-time performance; in Phillips & Brown (1999), the unilaterally- lesioned animals took longer to respond, however this effect was to the ipsilateral side only. Phillips & Brown (1999) replicated their findings in a subsequent study (Phillips & Brown, 2000), and found that unilateral STN lesions do not change reaction times, nor impair accuracy or error rate in a nose-poke reaction time task, but that unilateral lesions increase the likelihood of premature responses, compared with control surgery. The above evidence – from both clinical populations, and experimental animals – suggests that there is a qualitative and quantitative difference between unilateral stimulation/lesioning of the STN and bilateral. In humans, bilateral HFS for Parkinson’s patients may lead to more reliable improvements in motor dysfunction, but concurrently introduces more severe impairments in cognition. It is worth noting that the therapeutic response to bilateral or unilateral HFS may partially be determined by the asymmetry of the disease (i.e., which hemisphere of the brain is more affected by Parkinson’s disease); in cases where the severity of Parkinsonian degeneration is unilateral, STN-HFS of the contralateral hemisphere may improve therapeutic outcome (Hershey et al., 2008). In rats, unilateral lesions yield a weaker effect on cognition compared with bilateral lesions; bilateral lesions to the STN introduces a variety of cognitive deficits not expressed following unilateral lesions. Therefore, for the present experimental chapter with only one bilaterally lesioned rat, it is most likely that the majority unilaterally-lesioned animals retained sufficient cognitive function to complete the task with minimal impairment.

3.4.2 Conclusions

In summary, predominantly unilateral STN/ZI lesions were insufficient to produce pronounced behavioural deficits on the 7-stage task and the modified 11-stage ID/ED task. There was a suggestion that the unilateral lesions may have induced mild cognitive impairment, but in the absence of robust evidence of set formation in control rats, further research is needed, with a larger sample of bilaterally lesioned rats, before conclusions can be drawn on the explicit contribution of the STN to attentional set-formation.

105

Chapter 4

Using a palatable jelly tablet to deliver cognitive-

enhancing drugs in an ‘early/late probe’ task

Background:

This chapter presented an alternative behavioural approach; a more succinct alternative to the 11-stage task to infer the formation of attentional set, which provided the opportunity to measure the effects of putative cognitive-enhancing drugs.

Methods:

The first experiment examines the efficacy of a reduced-stress jelly tablet to deliver modafinil, in which brain concentrations and effects on locomotor activity are measured. The second experiment tested STN-lesioned rats on an ‘early-late’ probe task, with or without cognitive-enhancing drugs. The task inferred set by comparing two probe stages; one before set is likely formed, and one after multiple ID stages.

Results:

Brain concentrations of modafinil were comparable to oral gavage and modafinil, as expected, sustained locomotor activity, but only for later time-periods. Histology results for experiment II were consistent with Chapter 3; incomplete lesion damage minimised differences between groups, also drugs did not have an effect.

Conclusions:

Owing to an ongoing inability to visualise lesion damage, which may have attenuated the effects of the drugs, we cannot measure the role of the STN in set-formation from the current experiment. A refinement in how the STN is manipulated is pertinent.

106