The median preoptic nucleus (MnPO) of the lamina terminalis receives multiple afferent inputs from neuroglial structures of the hypothalamus, pons and medulla, regarding tonicity and volume of the extracellular fluid (ECF) compartment as well as the thermal status of the animal. Efferent projection of MnPO neurons to the parvocellular section of the paraventricular nucleus (pPVN) represents the main pathway to finally integrate signals of various homeostatic control loops. Nitric oxide (NO) has proven to act as prime neuromodulatory candidate underlying hypothalamic control of both body temperature and ECF homeostasis, and neuronal NO synthase (nNOS) is expressed in numerous MnPO neurons. The aim of the presents study therefore was to clarify whether nitrergic MnPO neurons projecting to the pPVN are responsive to systemic thermal, osmotic and volume stimulations in the conscious rat.

Materials & Methods: 

Adult male rats were used for retrograde neuronal tracing of MnPO neurons projecting to the pPVN by stereotaxic application of True Blue into the pPVN. Body core temperature, locomotor activity and water intake of rats were measured by telemetry devices in euhydrated animals (EUH), animals subjected to water-deprivation for 24 h (DEH), short-term acclimation to 30°C for 48 h (HEAT) or isotonic hypovolemia due to polyethylene glycol treatment (PEG). In coronal, perfusion-fixed brain sections, double immunohistochemistry for nNOS and Fos (marker of neuronal activation) was combined with the fluorimetric detection of retrograde labeling to quantify the number of activated (non-) nitrergic neurons projecting to the pPVN. Functional activation of (non-)nitrergic MnPO neurons was evaluated employing a primary Fura-2 loaded MnPO cell culture system and stimulation of MnPO neurons with angiotensin II (AngII) as an important neuromodulator relaying changes in ECF tonicity to MnPO neurons.

Results & Conclusions: 

Telemetric data combined with blood analysis allowed to define DEH, HEAT and PEG treatments as rather pure osmotic, thermal and volume stimuli. All stimuli evoked significant nuclear Fos translocation in MnPO neurons, with comparable numbers of Fos immunopositive cells in various MnPO subunits, but differential pattern formation depending on the type of stimulus applied. Osmotic and volume, but not thermal stimuli led to marked activation of MnPO-intrinsic nitrergic neurons (10–25%), indicative of NO being involved in MnPO mediated perception and/or integration of afferent osmoregulatory signals. Some15–20% of these activated NOergic neurons projected efferently to the pPVN. Functional data obtained in a primary MnPO cell culture system using the intracellular calcium imaging technique demonstrated activation of nitrergic neurons exclusively (100%) by AngII, a known peptidergic neuromodulator of osmosensitive subfornical organ neurons outside the blood-brain barrier, that efferently innervate MnPO intrinsic neurons.