Dr. Wolfgang Walz

Professor and Department Head
Department of Physiology
Dipl.-Biol (1977)Universitat Konstanz
Dr. rer. nat. (1981)Universitat Konstanz

Biology and Pathology of Astrocytes

Chloride Ions as Intracellular Modulators of Ion Channels

Using astrocytes in situ and in vitro, we found direct evidence for internal chloride changes to modulate the A potassium current, but not the delayed rectifier current. The GABAA receptor agonist muscimol has a dual effect: it opens a chloride channel, which causes in astrocytes an efflux of chloride. This transient decrease of the chloride concentration causes an inhibition of the A current. Using anions of the Hofmeister series, we find an order of potency of: chloride < bromide < iodide, which is the order of decreasing charge density. A dose-response curve reveals that the currents are most sensitive to changes around the physiological chloride concentration. We find that the inhibition of the A current with decreasing internal chloride concentrations is based on a depolarizing shift of the steady-state activation, but not inactivation. The results are compatible with the interference of these anions with the surface potential, which will result in an additional bias of the voltage sensor, as suggested by other authors finding similar actions of chloride in other preparations.

The hypotheses to be tested are:

• Intracellular chloride ions act as modulators of ion channels in muscle cells and neurons.
• They exert their effect by alterations of the membrane surface potential, in a way that the bias of the voltage sensor is shifted.
• In cell types, that exhibit large fluctuations of their internal chloride concentrations, this mechanism has physiological and pathophysiological significance.

We are using four different preparations to test these hypotheses:

• HEK 293 cell line with the expression of the following subunits in various combinations: Kv1.4, Kv4.2, Kv1.5, Kvß1.3. Whole cell and single channel studies will be performed to study channel kinetics. The external and internal chloride concentration as well as the anions will be varied.
• Three neuronal in situ preparations will be used, each with a different chloride gradient: adult CA1 pyramidal cells (active Cl extrusion), neonatal CA1 pyramidal cells (passive distribution) and cerebellar granule cells (active accumulation). L-type calcium, A-type and delayed type potassium whole-cell currents will be analyzed for their kinetics in internal solutions with different anions.
• The L-type current of cultured astrocytes will be analyzed with perforated whole-cell patch and different internal anion concentrations.
• We will use isolated aortic smooth muscle cells and isolated aortic rings to prove that adrenergic agonists change the internal Cl concentration, which in turn will modify calcium current(s) to change the force of contraction.
• We assume these data will verify the role of chloride for a variety of ion channels. We also think the anticipated compatibility of the results with the anion order of the Hofmeister series and a similar bias in voltage-sensitive processes will strengthen the argument for a mechanism of action that involves the surface potential.

Reactive Gliosis in Focal Ischemia

We are interested in the gliotic tissue or glial scars in the brain, its genesis and control, and its role in the injured brain.

Functional Specialization of Components of Reactive Gliotic Tissue
This project is based on the previous (electrophysiological and immunocytochemical) work in my laboratory on the following preparations:

• Different subtypes of normal astrocytes in situ.
• Reactive astrocytes in the gliotic hippocampus after diffuse neurotoxic injury
• Three distinct subpopulations of reactive astrocytes after focal ischemia

We continue the last subject by using the pial vessel disruption model, a cortical devascularizing injury type, to investigate the properties of these three distinct populations of reactive astrocytes. We will use a combination of in situ electrophysiology and immunocytochemical methods and manipulation of the inflammatory reaction accompanying the injury. Three distinct reactive astrocyte populations were identified:

• A 200 µm thick boundary layer (eight cells wide) of S100+/GFAP-/VIM- cells surrounds the lesion. After three weeks the lesion develops into a cystic cavity, by that time the boundary layer transformed into a GFAP+ glia limitans.
• At the base of the lesion there is a large area that develops into 100+/GFAP+/VIM+ cells and contains proliferative cells. It therefore has to be regarded as the local reactive astrocyte.
• Another layer surrounds the boundary and base that is 100+/GFAP+/VIM-. It therefore has to be regarded as the remote reactive astrocyte.

We investigate dye coupling and membrane currents as well as the appearance of nestin, laminin, CNTF and proteoglycan in all of the three subtypes. In the layer of the boundary astrocyte we correlate these factors with the appearance of a leptomeningeal cell layer and of GFAP. We will use injections of minocycline to see if this anti-inflammatory drug is causing any changes to the boundary layer and its properties. This would give an indication that inflammatory processes are controlling major features of the boundary astrocyte.

A similar approach is used for the local gliotic response. These factors are correlated with the loss of the neuronal elements. A variation of the surgical procedure (2 mm instead of a 5 mm borehole) leads to a more shallow injury and no significant inflammation or boundary layer. This will allow us to investigate which properties are upregulated by the presence of inflammatory processes and which are dependent on neuronal loss only.

The remote gliotic response is also investigated, similarly to the local response. We expect the presence of CNTF but not of nestin and proteoglycan. The manipulation of the inflammatory processes should not change the properties of this remote response, except its extend. We will initiate in normal rats spreading depression waves and expect to find S100+/GFAP+/VIM- cells with similar properties than the remote reactive astrocytes. Since spreading depression waves alone have a protective effect on subsequent injuries, this would be an indication of a major role of spreading depression in the genesis of a remote reactive astrocyte type with mainly protective function. We expect that the local reactive astrocyte has much more pronounced inhibitory molecules (proteoglycan) in its extracellular matrix and that this detrimental property is induced mainly by inflammatory processes.

We would be in a position to characterize the functional properties of these three distinct and segregated populations. We would be able to identify benefical and detrimental properties on neuronal survival.

Minocycline as a Protective Anti-inflammatory Agent
In this study we use the mouse in conjunction with the pial vessel disruption model. We want to manipulate the inflammatory response of the gliotic tissue in order to show that the presence of the vimentin positive local reactive astrocyte and of proliferative reactive astrocytes depends on inflammatory processes. Minocycline is a tetracycline derivative, which has been shown to be neuroprotective and to act on inflammatory processes. We use the above approach to investigate if minocycline treatment is reducing neurophil invasion, lesion size and boundary layer as well as the extent of BrdU positive S100+/GFAP+/VIM+ cells and their surface and membrane properties. The results will be correlated within the framework of the Canadian Stroke Network with other minocycline dependend projects on microglia, neutrophils, lesion volume and behavioural recovery.

Contact Information: Department of Physiology, University of Saskatchewan 107 Wiggins Road Saskatoon SK S7N 5E5 CANADA       Phone:  306-966-6532  Fax:      306-966-6530  Email:    walz@sask.usask.ca