Anxiety induces behavioural and structural changes in neuronsZoe HancoxLife sciences

Introduction

Anxiety disorders are a part of a wide spectrum of disorders, which represent the most common of the psychiatric diseases. They include panic disorders, specific phobias, social phobias and posttraumatic stress disorder (PTSD). In 2005, Kessler et al carried out an epidemiological study which found that the lifetime prevalence of any anxiety disorder is 28.8%, yet there are few effective treatments available.One of the brain regions thought to be involved in fear and anxiety is the central nucleus of the amygdala (CeA) as it is in a strategic position to mediate many aspects of fear and anxiety behaviours. There were two aims to this project. The first was to determine if animals respond to an innate stressor is an all-or-nothing type response or graded in a dose-dependent manner. The second aim was to determine whether plastic changes occur in neurons of the CeA, as these changes are linked to long-term emotional memory, which may underpin phobias and PTSD.The project utilised a number of techniques to investigate fear, including: a behavioural model of innate fear, immunoassays and histological analysis.Ref: Kessler et al (2005), Archives of general psychiatry, v. 62, no. 6. 2005.

MethodsAll procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines and under local ethical approval.

Behavioural runs. Rodents were placed in behavioural boxes containing a red hide area (size: 100x75x67mm, entrance: 40x35mm), which was constructed from red Perspex (allowing clear visualisation of the interior under red light illumination). Animals were habituated to the boxes for 2 days prior to testing. Cotton strips (7x70mm) were positioned on the back wall of the hide section, 10 mm from the floor. On test days; animals were exposed to one of two strips: control (no odour, C), or cat-odour impregnated (PSS) strips. For the control and predator scent only runs a strip of 7x70mm was used. To determine whether exposure to PSS evoked dose dependent changes additional runs were performed using small strips (3.5x70mm, 0.5xC/PSS) or big strips (21x70mm, 3xC/PSS).Analysis. Various behaviours were analysed from the recorded behavioural runs.Golgi staining. Tissue was obtained and following a two week impregnation period the tissue samples were embedded in 4 % agarose and 200 µm sections were cut. Slices were mounted on gelatine-coated slides and processed for visualisation, according to manufacturer’s instructions and solutions provided in the FD Rapid GolgiStain™ kit.Spine counts. Tissue slices were viewed under microscope and recordings were made of bitufted or stellate cells present in the CeA.ELISA. Using the plasma samples collected from the rodents an ELISA was carried out to determine the concentration of corticosterone. The procedures set by the manufacturer, Arbor Assays, were followed.

Figure 1 . The amount of time spent digging on the test days. There is an increase in the amount of time spent digging by rodents exposed to predator scent big strip, compared to rodents exposed to the predator scent small strip. This data is statistically significant.

Figure 2 . The amount of time spent grooming on the test days. The exposure of the predator scent big strip caused a decrease in the amount of grooming rodents carried out, compared to both the control and predator scent small strips. This data is not considered statistically significant.

Figure 3 . The amount of time spent rearing on the test days. There is a small increase in the amount of time spent rearing by the rodents exposed to the big strip, compared to those exposed to the small strip. This data is statistically significant.

Figure 4 . The amount of time spent in the behavioural box on the test days. Compared to the predator scent infused small strip, the predator scent infused big strip caused an increase in the amount of time spent in the box. This data is not considered statistically significant.

Figure 5 . The number of transitions from the light to the dark (hide area). The presence of the predator scent infused big strip caused an increase in the number of transitions, compared to the predator scent infused small strip. This data is not considered statistically significant.

Figure 6. The number of times the rodent made contact with the strip. This figure demonstrates that the presence of the big strip, either control or predator scent, caused an increase in the number of times it was contacted, compared to the small strip. This data is not considered statistically significant.

Figure 7. The amount of time spent making contact with the strip. The presence of the big strip caused an increase in the amount of time spent contacting the strip, compared to the small strip. This data is not considered statistically significant.

Results

Figure 8. The corticosterone concentrations of the rodents. The predator scent infused big strip caused a increase in the corticosterone concentration, compared to the predator scent infused small strips. This data is not considered statistically significant.

Figure 9. (a) The CeA (circled) at 4x magnification. (b) A schematic representation of the positioning of the CeA (circled). (c) A spiny neuron from the CeA. (d) The spine count from neurons of the CeA. There appears to be little difference in the numbers of spines recorded between the predator scent infused big and small strip, indicating there is no plastic changes occurring. This data is considered non significant.

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ConclusionIt appears that the presence of predator scent infused big strips does alter the behavioural response, compared to the predator infused small strips. This indicates that the behavioural response to predator scent is dose dependent. However due to some of the data not being considered statistically significant more work will need to be carried out to prove this theory. Nevertheless some of the behavioural analysis data was found to be significant and therefore there is some indications that the response is dose dependent. It was found that there was no plastic changes occurring in the CeA, but as the data was not considered statistically significant it will have to be further investigated further.

AcknowledgementsSpecial thanks to my supervisor Dr Dawn Collins and Angharad Jones for their help and guidance. Also a mention to the researchers in the Neuroscience department and laboratories for their help.

Histological analysis of spine density

Results of Corticosterone Assay

Behavioural Testing

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