Publication Date

2009-07-28

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Physiology and Biophysics (Medicine)

Date of Defense

July 2009

First Committee Member

Stephen Roper - Committee Chair

Second Committee Member

Charles Luetje - Committee Member

Third Committee Member

Michael Norenberg - Committee Member

Fourth Committee Member

Gerhard Dahl - Committee Member

Fifth Committee Member

Kenneth Muller - Mentor

Sixth Committee Member

Eliana Scemes - Outside Committee Member

Abstract

ATP released upon nerve injury is an important chemotactic activator of microglia; however, the source of the extracellular ATP is uncertain. In large glial cells such as astrocytes, chemical or mechanical stimulation or injury causes an intracellular calcium wave that moves from cell to cell propagated by extracellular ATP through a regenerative ATP-induced ATP-release mechanism. In mammals a prime candidate for the ATP release channel is the pannexon, a hexamer of the invertebrate gap junction homologue pannexin1, although the pharmacologically similar protein connexin is also a candidate. I hypothesized that upon nerve injury, ATP released through pannexon channels activates microglia and initiates their movement. My experiments were done on leeches (Hirudo sp.) in part because they lack connexins, their microglia are regulated by ATP, and their glia are especially large and produce calcium signals. I found that in the giant glia of leeches the 2 homologues of pannexin1, innexins2 and 3 (HmINX2 and HmINX3), formed innexons with similar properties to pannexons. Innexons opened and released ATP upon depolarization or in elevated extracellular potassium solution, were permeable to dye molecules, and were blocked by carbenoxolone (10 to 30 µM) or saturated CO2. Pharmacological experiments showed these channels were essential for the migration of microglia to nerve lesions, but calcium waves in single leech glia did not require innexons or exogenous ATP. In addition, nerve injury typically produces arachidonic acid, which closed the innexons and thereby blocked microglia migration. The results were consistent with those in the leech showing that ATP activates microglia and NO is important for their directed movement. The contribution of these findings to a general understanding of the molecular signals regulating the activation of microglia is discussed.

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