Date of Award
Doctor of Philosophy (PhD)
William E. Armstrong, Ph.D.
Joseph C. Callaway, Ph.D. Robert C. Foehring, Ph.D. Detlef Heck, Ph.D. Fuming Zhou, Ph.D.
Magnocellular neurosecretory cells (MNCs) are large oxytocin (OT)- and vasopressin (VP)-releasing neurons that secrete these hormones into the circulatory system in response to physiological stimuli. These cells exhibit unique phasic and burst firing patterns to release these peptides into the circulatory system where they primarily control milk ejection and parturition (OT) as well as salt-water balance and vasoconstriction (VP). This firing is underlain by intrinsic ionic mechanisms that shape the duration and frequency of these bursts. One of these mechanisms is the Ca2+-dependent afterhyperpolarization (AHP), which activates during bursts and causes spike frequency adaptation. This afterhyperpolarization has three distinct conductances: a fast component (fAHP) underlain by BK channels, a medium component (mAHP) underlain by apamin-sensitive SK channels, and a slow component (sAHP) which is an apamin-insensitive K+ conductance. The mechanisms that control the sAHP are poorly understood in MNCs. The work embodied here explores the mechanisms involved in generation of AHPs, specifically how the phospholipid, PIP2 can activate and modulate the mAHP and sAHP. The major discovery is that the mechanisms that generate mAHP and sAHP are different between OT and VP neurons. PIP2 depletion via wortmannin in the cells abolishes the mAHP and sAHP of OT but not VP neurons. This demonstrates OT neurons require PIP2 to activate an AHP while VP neurons do not. Interestingly, increasing PIP2 within the cells has little effect on OT neurons while drastically enhancing the sAHP in VP neurons, thus PIP2 plays a different role in both cell types. In OT neurons, PIP2 exerts its effect by facilitating Ca2+ entry through voltage-gated Ca2+ channels, demonstrated by inhibited Ca2+ currents in the presence of wortmannin.
The mechanistic differences extend to which Ca2+ channels contribute Ca2+ to the mAHP and sAHP. In OT neurons, N-type Ca2+ channels couple primarily to both components. In VP neurons, N-type channels couple to the mAHP while the sAHP receives a contribution from R-type channels. The precise way PIP2 modulates Ca2+ channels in OT neurons is explored further in dissociated neurons genetically labeled for OT or VP. PIP2 depletion inhibited the amplitude, shifted the steady-state activation curve leftward, and modestly accelerated the inactivation of both the whole-cell and isolated N-type current in OT neurons only. This suggests that PIP2 is not required, but is a co-factor, for channel activation. The PIP2 mechanisms of AHP modulation in VP neurons appear complex, as the enhancement observed during increased PIP2 didn’t occur when EGTA was replaced with fura-2 in the pipette. In order to understand what happens to [Ca2+]i during this enhancement, we changed the Ca2+ indicator to fluo-4 and was able to observe enhancement under specific conditions. This suggests that VP modulation is critically dependent on the time course and buffering of available Ca2+.
Finally, I also performed a cursory evaluation of possible morphological implications in AHP generation. I used regression analysis to assess the relationships between AHP amplitude, [Ca2+]i, and dendritic size. There is a moderate relationship between AHP amplitude and dendritic length in both OT and VP neurons, suggesting that a considerable portion of the AHP conductance could occur in the dendritic tree of these neurons. These studies highlight the unique AHP mechanisms between OT and VP neurons of supraoptic nucleus.
Kirchner, Matthew Karl (http://orcid.org/0000-0001-6960-7685), "PIP2 Modulation of Afterhyperpolarizations in Magnocellular Supraoptic Neurons" (2018). Theses and Dissertations (ETD). Paper 479. http://dx.doi.org/10.21007/etd.cghs.2018.0460.
Available for download on Saturday, August 21, 2021