Online

Kolowrat, Samantha

Pharmacology

2021

M.S.

The paraventricular nucleus of the hypothalamus (PVN) plays a critical role in regulating the hypothalamic-pituitary-adrenal (HPA) axis, sympathetic activity, cardiovascular system and stress responses.1,2 This brain region is primarily controlled by GABA and glutamate inputs from the peri-PVN region.3 Two significant modulators of these GABA-glutamate systems are Angiotensin II (Ang II) and brain derived neurotrophic factor (BDNF). It has been postulated that BDNF and Ang II signaling rely on each other via cross-activation of their receptors and by activation of the non-selective cation channel, the transient receptor channel type C3 (TRPC3).This thesis aims to study changes in intracellular calcium levels as an indicator of neuronal activity in the PVN, and to investigate the role of Ang II, BDNF and TRPC3 mediated mechanisms. Five-week-old rats were injected with AAV2-CaMP6f to visualize intracellular calcium in PVN neurons with a multi-photon microscope. We developed tools to analyze changes in calcium signals, which employed mathematical algorithms to determine regions of interest (ROIs) based on variability of GCaMP6f fluorescence. Calcium signals were analyzed using the number of active sites, or ROIs, average intensity and cumulative intensity of all ROIs within the field of view. Time control experiments demonstrated that neurons in our preparation displayed "spontaneous" activity and were not significantly affected by prolonged laser exposure. In addition, we found that inhibition of sodium channels with TTX, administration of GABA and the L-type calcium channel inhibitor nifedipine significantly blocked PVN neuronal calcium events. An inhibitory cocktail of biciculline (GABAA antagonist), AP-5 (NMDA receptor antagonist) and CNQX (AMPA receptor antagonist) was used to block peri-PVN input, and overall decreased the number active sites present, while the average intensity was unchanged. We found no significant changes in our measured parameters in response to Ang II, activation of the BDNF receptor and activation or inhibition of TRPC3 channels. These results demonstrate the potential of this new method to study and analyze calcium changes in PVN neurons. We were able to show that neuronal activity can be successfully inhibited by TTX, GABA and nifedipine. However, the role of TRPC3 and the interaction between BDNF and Ang II remains unclear.