This is a application proposes to study migration of mast cells into the medial habenula of ring doves during courtship. Previously proposed studies on transplantation and calcium imaging have been eliminated, and others have been reduced in scope. The application consists of two major specific aims. In the first aim, experiments will determine when mast cells in the medial habenula degranulate during the reproductive cycle, and/or following administration of gonadal steroids, and whether the blood-brain barrier is altered in response to mast cell degranulation. They will also determine what triggers mast cell secretion in vitro, especially whether substance P is involved, and whether mast cell products alter channel properties of habenular cells in vitro or synaptic responses to input from cocultured septal explants. In the second major aim, the mechanisms that determine mast cell number will be addressed. Experiments are proposed to determine whether mast cells divide in the habenula, migrate from outside, have a special relationship to astrocytes and whether habenular astrocytes or neurons express particular chemokines or matrix molecules which may allow this migration. In vivo studies of blood vessel permeability during mast cell migration will be conducted. The state of mast cell degranulation will be assessed using point counting stereology from electron micrographs. The role of neuropeptides (substance P) and steroids in regulating histamine, ATP, and prostaglandin secretion will be examined in dissociated medial habenular cultures. Histamine, ATP, and prostaglandins will be assayed in the medium. Alterations in neural activity and synaptic transmission will be examined in medial habenular neurons cocultured with septal explants. 3D reconstruction of mast cells will be used to determine what cellular elements they are associated with during the reproductive cycle. Factors unique to the medial habenula which allow mast cell migration into the structure will be assessed. Alterations of the blood-brain barrier following mast cell degranulation will be assessed using Evans blue fluorescence. Regions outside the blood-brain barrier, i.e. the circumventricular organs, will be used as a positive control. Paraventricular and supraoptic nuclei will be used as a negative control (contain no mast cells and are inside the blood-brain barrier but are well vascularized).
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