Development of a Novel Method to Assess the Effects of Predictability and Chronic Stress on Neuronal Morphology and Decision-Making in Rats

The Golgi-Cox stain remains the gold standard for studying changes in neuronal morphology offering the greatest detail and clearest spine visualisation. Nevertheless, the method has limitations particularly in thick sections where laser penetration, inadequate 3D cell reconstructions, background staining and inadequate cell visualisation within fixed or otherwise non-fresh tissue offer challenges to the microscopist. Here I describe the development of a more efficient, cost effective and more broadly applicable stain together with my attempt to apply this new stain to modern tissue clearing techniques. Not only did the new methodology improve the staining of cells, it enhanced CLARITY and CUBIC clearing techniques allowing the clearing of brain tissue within a fraction of the usual time. Having developed this new methodology, I then applied this new approach to study the changes in neuronal morphology induced by chronic stress in rats. Changes in morphology in a large number of brain regions were analysed and relate to the functional effects of chronic stress. Chronic stress has been repeatedly shown to change morphology in profound ways, and I observed both increases and decreases in dendritic lengths and spine densities in different brain regions. In addition to the morphology analysis, I investigated the concomitant effects of chronic stress on choice and decision-making in the instrumental conditioning situation. Chronically stressed rats presented with decreased sensitivity to changes in the value of the instrumental outcome and in the action-outcome contingency. Furthermore, chronically stressed rats expressed a facilitation of outcome specific Pavlovian-instrumental-transfer (sPIT). The ability to control or to predict the application of stress has been reported to ameliorate its effects and, in a subsequent experiment, I compared the effects of random chronic stress and predicted chronic stress. Consistent with the previous literature, when the rats could predict the application of stress, its detrimental effects were reduced. Although the effects of random chronic stress were similar to those observed in the previous experiment, rats exposed to predictable stress showed sensitivity to changes in outcome value and in the action-outcome contingency comparable to unstressed controls. Nevertheless, in other tests of decision-making predictably stressed rats showed a further facilitation in the sPIT effect together with a deficit in delayed discounting. These changes in decision-making were correlated to changes in neuronal morphology caused by chronic stress. Again, randomly stressed rats presented with degenerated dendritic length and synaptic spine density across many subregions of the prefrontal cortex, and proliferated morphology within the nucleus accumbens core and basolateral amygdala (BLA). Consistent with the idea that predicted stress would protect the animal from the detrimental effects of chronic stress, predicted stress rats expressed as controls within the dorsomedial striatum (DMS), hippocampus and medio-orbitofrontal cortex (MO), along with other morphology changes not consistent with random stress rats, such as a proliferation of the BLA more so than controls but not as much as random stress rats. Of the many arguments made, principally we propose that the protection of the DMS and MO and their crucial involvement in goal direct action, explains why, unlike random stress rats, predicted stress rats maintain sensitivity to outcome devaluation and contingency degradation. These morphological changes were analysed with the newly developed ultra-rapid Golgi (URG) stain. Which with further development and use of two-photon microscopy has been designed to excite stained neurons to auto-fluoresce. The two-photon laser was manipulated to excite the mercury compounds impregnated into the cells, energising the mercury to the point of electron displacement, this is a completely novel visualisation of Golgi stained neurons, offering even greater visual clarity and analysis