Morphological Study On Response Of Astrocytes In Rat Brain To Change Of Osmotic Pressure And Relationship With Neurons

For a long time, astrocytes have been thought to provide mainly a structural, trophic, and metabolic support to neurons. With the development of research techniques, a growing body of evidence has emerged on the existence in the brain of a close bidirectional communication between neurons and astrocytes. Astrocytes are not inertia cell and can regulated actively neuronal activity.Objective: In this study we try to simultaneously investigate the response of neurons and astrocytes of rats following hyperosmotic stimulation and focus attention on the role of neuronal-astrocytic complex in the osmotic reglution.Methods: Hyperosmotic pressure animal model was established by administering 3% sodium chloride as drinking water to rats or increasing osmotic pressure of the culture medium. Osmoregulation positions in the brain, reciprocal projection pathways between the medullary visceral zone(MVZ) and supraopticnucleus (SON) or hypothalamic paraventricular nucleus(PVN), oscillation of intracellular calcium in cultured neurons and astrocytes were studied by means of anti-Fos, glial fibrillary acidic protein (GFAP), tyrosine hydroxylase (TH) or vasopressin (VP) multiple imrnunohistochemical staining, immuno-electronic microscope, WGA-HRP retrogradely tracing and cell culture methods.Results: (1) Fos positive neurons within the MVZ, parabrachial nuclei, locus ceruleus, PVN,SON, subfomical organ increased markedly. There were also a large number of GFAP positive structures in the brain and their distribution pattern was fundamentally similar or analogous to Fos positive neurons in the above-mentioned areas. The augmented GFAP reactivities took on hypertrophic cell bodies, thicker and longer processes. Triple immunofluorescence histochemical staining showed that a neuron could be closely surrounded by many astrocytes and they formed neuron-astrocytic complex (N-ASC). Fos+/TH+/GFAP+ and Fos+/ VP+/GFAP+ triple labeled N-ASC could be found in the MVZ, PVN and SON respectively; (2) Under electronic microscope, the astrocytic processes connected closely with the dendrites or axons of the neurons, where the bilateral membranes became thick. We call transiently it electron-dense areas (EDAs). The number of EDAs increased remarkably following hyperosmotic stimulation; (3)When trace retrogradely, WGA-HRP was microinjected into the unilateral SON, PVN or nucleus of solitary tract (NTS) respectively using the stereotaxic method, the N-ASCs formed by the neurons triple-labeled with HRP/Fos/TH (or VP) and astrocytes labeled with GFAP could be found in the MVZ, SON and PVN respectively; (4)After being treated with heperosmotic NaCl solution, intracellular calcium concentration in cultured hypothamic neurons and astrocytes increased and then decreased. At last, it came to a little higher level of resting intracellular cacium. The velocity of astrocytic intracellular calcium elevation to the peak value is a slightly faster than neurons.Conclusions: The present results indicated that the brain areas involving in the heperosmotic regulation mainly located in MVZ SON and PVN. The neurons andastrocytes might be very active following hyperosmotic pressure and N-ASC as a functional unit might serve to modulate osmotic pressure. There was reciprocal osmoregulation pathways between the MVZ and SON or PVN in the brain.