George G. Somjen
Professor Emeritus of Physiology in Cell Biology

Hypoxia of brain tissue.
For the last 15 years our laboratory studied the transient effects of brief hypoxia, as well as the mechanisms of lasting damage caused by oxygen deprivation in mammalian brain tissue. We are using live tissue slices freshly prepared from rodent brain. The most frequently used preparation is rat hippocampus, but we have also experimented with mouse spinal cord and gerbil hippocampus.

Some of the more important findings of past years include:
Synaptic transmission rapidly fails in hypoxic brain and spinal cord, because presynaptic calcium channels become incapable of opening and, as a result, transmitter cannot be released by presynaptic impulses. Upon timely reoxygenation this effect is readily reversible.

One of the major factors causing irreversible neuronal failure during hypoxia, is the prolonged elevation of intracellular calcium. Calcium enters neurons from interstitial fluid because of a large increase of membrane conductance.

pH effects on neurons.

One of the consequences of cerebral hypoxia is tissue acidosis. Acidosis and alkalosis are frequent clinical problems in other conditions as well. We studied the effects of pH on neuron function within the clinically relevant pH range. We have found that mild to moderate acidosis improves recovery of neuron function from transient hypoxia. By this mechanism brain tissue is able to protect itself to some degree against hypoxic damage.

Acid pH of a degree that can occur in human patients suppresses voltage dependent potassium, sodium and calcium currents in freshly isolated hippocampal neurons. Calcium currents are more strongly affected than the other currents. Alkaline pH enhances these currents.

Intracellular pH changes modulate Ca currents and, by this mechanism, alter neuronal responses to stimulation.

Osmotic effects on brain tissue and on isolated nerve cells.

One of the consequences of tissue hypoxia is cell swelling. To assess the degree to which neuron swelling contributes to hypoxic neuron impairment, we are investigating the effects of changing extracellular osmotic pressure on brain tissue and on isolated neurons.

Increase of osmolarity which causes cells to shrink depresses synaptic transmission in brain tissue slices and it also suppresses voltage dependent ion currents of isolated neurons. Elevated osmotic pressure dramatically improves the chances of recovery of neuron function following transient hypoxia. Such hypertonic treatment also prevents neuron depolarization and the associated entry of calcium into cells.

Lowering of osmolarity of brain tissue slices causes cell swelling and it enhances synaptic transmission. Excitatory synaptic currents of hippocampal neurons are strongly enhanced, inhibitory synaptic currents are less strongly affected. Lowering of NaCl concentration without osmotic change causes similar but weaker effects. Prolonged exposure to strongly hypotonic solution causes lasting impairment of synaptic transmission, but does not kill neurons.

In collaboration with Dr. W. Wadman's laboratory in Amsterdam, calcium activity in neurons in hippocampal tissue slices and in freshly isolated neurons was measured with the Ca-sensitive fluorescent indicators. Similar experiments were also conducted using the shared departmental confocal microscope. Hypotonic treatment caused an initial transient decrease, followed by a sustained increase of intracellular calcium. Low NaCl had similar if weaker effect.
Imaging of neuron volume changes.

In collaboration with Dr. Dan Kiehart have recorded the degree to which the size and shape of isolated neurons changes under the influence of osmotic force. In a parallel study a different technique was used for the same purpose in the laboratory of Dr. Wytse Wadman in Amsterdam.

Three types of behavior were seen when isolated brain cells were exposed to hypotonic environment: slow swelling; delayed swelling; and resistance (i.e. no swelling even after 30 min treatment). Hypertonic treatmemnt caused slow shrinkage in most cells.

Imaging of intrinsic optical signals of brain tissue slices.

In collaboration with Dr. Dennis Turner of the Division of Neurosurgery, we are obtaining computer-generated images of changes in transmitted or reflected light of hippocampal tissue slices during hypoxia, spreading depression, and osmotic changes.

Marked differences in regional distribution of optical responses were registered. Most surprisingly, the same treatment (hypoxic SD-like depolarization) can cause either increase ordecrease of translucence of the affected area of the tissue.

The depolarization caused by tissue hypoxia spreads in the tissue in a manner similar to "classical" spreading depression (SD).

Mechanism of spreading depression.

The role of gap junctions in the propagation of spreading depression (SD) was
investigated. Treating hippocampal slices with drugs that are known to block gap junctions reversibly prevented SD propagation.
New, ongoing projects (1997)

The effect on hypoxic SD-like depolarization of a variety of drugs with known action on membrane transport proteins is being studied.
Single channel currents are recorded from neurons in tissue slices during SD and SD-like depolarization.

The effect of pH changes on synaptic activity is analyzed using whole-cell patch-clamp recording in neurons in tissue lsices.

Membrane ion currents and cell size and shape changes are recorded simultaneously, in isolated neurons, using patch-clamp electrical recording and confocal microscopy.
KEY WORDS
Electrophysiology; pathophysiology; stroke; brain hypoxia; seizures; ion channels;
neuron pH; acidosis; alkalosis; osmotic effects; cell swelling.

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