CENTRAL NERVOUS SYSTEM REGULATION OF BODY FLUID HOMEOSTASIS.

Book excerpt: ABSTRACTThe central nervous system (CNS) is critical to the regulation of body fluid homeostasis. Specialized hypothalamic neurons localized to the lamina terminalis detect changes in plasma osmolality or sodium (Na+) concentrations; the CNS integrates these inputs and coordinates downstream homeostatic responses to restore extracellular fluid (ECF) sodium chloride (NaCl) concentrations and osmolality back to normal levels. The mechanism by which hypothalamic neurons detect changes in circulating plasma NaCl concentrations or osmolality is not known. Central infusion of hypertonic NaCl in rats stimulates thirst, increases sympathetic nerve activity, and raises plasma vasopressin levels. These homeostatic responses to increased plasma and cerebrospinal fluid (CSF) NaCl concentrations are attenuated by pretreatment with the non-voltage gated Na+ channel antagonist benzamil. Among other channels, benzamil targets the epithelial Na+ channel (ENaC). Although the ENaC contains three subunits, (, , and ), the ENaC subunit is expressed in the hypothalamic nuclei containing osmo-or Na+-receptors. Therefore, we hypothesize that the ENaC subunit may be a brain Na+-detector that is important in helping the CNS detect changes in body fluid homeostasis. The first aim of this thesis is to determine whether the ENaC subunit contributes to hypernatremia-induced thirst responses. This research question was investigated using novel mice generated with brain-specific deletion of the ENaC subunit (ENaClox/loxNestinCre). Behavioral experiments measuring cumulative water intakes demonstrated ENaClox/loxNestinCre mice had attenuated water intake in response to sc injections of hypertonic NaCl compared to control (ENaClox/lox) mice. In contrast, ENaClox/loxNestinCre and ENaClox/lox strains drank similar volumes of water in response to hyperosmolality and extracellular dehydration induced by subcutaneous (sc) injections of hypertonic mannitol and isoproterenol, respectively. This suggests that the ENaC subunit may be a brain- Na+ detector as it is critical for normal thirst responses induced by hypernatremia but not hyperosmolality or extracellular dehydration. Manipulation of ENaC-expressing neurons in the lamina terminalis is necessary for a comprehensive understanding of this potential brain Na+-detector. Recent new technologies, such as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), have transformed the neuroscience field with the ability to remotely manipulate subpopulations of neurons. The second aim of this thesis was to determine if DREADDs technology can be implemented in the lamina terminalis to manipulate body fluid homeostasis. This research question was investigated using behavioral thirst experiments, single-unit recordings, and in-vitro electrophysiology. Male C57BL/6 mice received a stereotaxic microinjection of the excitatory DREADDs construct, hM3Dq, into the subfornical organ (SFO). After viral infection, mice received sc injections of clozapine N-oxide (CNO) and cumulative water intakes were recorded. Post-hoc immunohistochemistry for the hemagglutinin (HA)-Tag confirmed that mice expressing the DREADDs construct in the SFO (termed SFO-mice) drank significantly more water and 0.3M NaCl in response to sc CNO compared to mice lacking DREADDs expression in the SFO (termed SFO-x mice). CNO also significantly increased the number of Fos-positive neurons in the lamina terminalis of SFO mice. In-vivo single-unit recordings demonstrated that intravenous (iv) injections of CNO increased the action potential firing rate of Angiotensin II (AngII)- or Na+-responsive SFO neurons, but not SFO-x neurons. Similarly, in-vitro patch-clamp experiments indicated that bath application of CNO produced membrane depolarization and increased neuronal discharge in SFO vs SFO-x neurons. Finally, chronic activation of SFO neurons, via administration of CNO in the drinking water, increased 24-hour water intakes in SFO, but not SFO-x mice. Therefore, DREADDs technology can be used to acutely and chronically manipulate SFO neuronal activity and alter body fluid homeostasis. With these results in mind, DREADDs technology can now be utilized to manipulate subpopulations of lamina terminalis neurons, such as ENaC-expressing neurons, to determine their significance in maintaining body fluid homeostasis. As a whole, the neuroscience field can utilize DREADDs technology to further investigate the neuronal mechanism underlying osmoregulation.