Maladaptive changes to Cholecystokinergic systems in the Periaqueductal grey in rats experiencing persistent behavioural disability in the wake of Neuropathic pain

In 2018, it was estimated that 3.24 million Australians were living with chronic pain, with the total estimated cost to the economy of $73.2 billion (Painaustralia, 2019). People with chronic neuropathic pain report substantial reductions in their quality of life, with disturbances to social relations; alterations to sleep, appetite, metabolic endocrine and sexual dysfunction; a loss of interest in external events; and moderate to severe depression. More often, it is these disabilities rather than the sensory disturbances of allodynia, hyperalgesia, and spontaneous pain, which are deemed by sufferers to be the most debilitating. The neural adaptations underlying the sensory changes of neuropathic pain have been studied in rats and, using a sciatic chronic constriction injury (CCI) model, in combination with resident-intruder behavioural testing, it has been elucidated that a subgroup of animals undergoes complex behavioural and physiological dysfunction despite all animals experiencing similar levels of pain (Kilburn-Watt et al., 2010, Monassi et al., 2003, Mor et al., 2011). The general aim of this thesis is to further characterise the complex behavioural and physiological disturbances (i.e. disability) in a subgroup of animals marked as having pain and persistent disability. Work from our group has focussed on a midbrain region, the Periaqueductal grey (PAG), as a site with the potential to contribute to behavioural disability. Some of this work used a molecular approach to assess gene expression patterns in PAG and identified a significant upregulation of mRNA encoding Cholecystokinin (CCK), a peptide neurotransmitter. Several brainstem and hypothalamic nuclei contain CCK-ergic cell populations, and these areas are also known to have reciprocal connectivity with the PAG. A pilot study (chapter 3) aimed to identify if rats which undergo stereotaxic surgery for retrograde tracing are compatible with the existing laboratory resident-intruder paradigm. We observed that, in animals that had undergone stereotaxic surgery and CCI, there was an increase in non-social behaviours compared with CCI-alone, though a larger sample size may negate this effect. Importantly, given the long time-frame of these experiments, we were able to successfully stain for both Cholera Toxin B (CTB) and CCK in both the brainstem and hypothalamus and found co-localization in the nucleus tractus solitarius and the hypothalamus. Populations of neurons within the PAG have distinct functions, as such it was an important step to determine where in the PAG these changes are taking place. With the use of in situ hybridization techniques Chapter 4 of this thesis showed significant up-regulation of CCK mRNA in cells of the ventrolateral PAG (vlPAG) and the adjacent nucleus of the dorsal raphe selectively in rats with pain and persistent disability, with these animals not only showing and increase in CCK mRNA expressing cells, but also indicating higher levels of CCK mRNA expression in these cell populations, with mean densities of silver grain labelling correlated strongly with the degree of behavioural change (decreased dominance) in resident-intruder interactions. This anatomical specific pattern of increased CCK expression in a functionally distinct subregion of the midbrain leads us to consider if similar changes were occurring in this region for the cognate receptors of CCK, the CCK1 and CCK2 receptors. Thus, in chapter 5, we aimed to see if the neurons of the midbrain PAG expressed these receptors, and to observe any differences in the expression of these receptors following CCI. Our findings showed that, following CCI, two distinct, behaviourally categorised groups of animals- non-affected and transient disability- display increased CCK receptor immunoreactivity, indicative of an adaptive response, whilst animals with pain and persistent disability showed significantly lower expressions of CCK receptors in functionally relevant regions of the PAG despite all animals showing identical levels of sensory disturbance characteristic of neuropathic pain. The PAG is an important integrator of pain and behaviour. A selective upregulation of CCK in animals displaying pain and persistent disability is of particular significance given that we have shown the juxtaaqueductal gray and the vlPAG receive inputs from areas of the brainstem and hypothalamus that are important not only in exerting descending inhibition of pain transmission, but also in the affective components of pain