The Department of Neuroscience is a joint institution with the Department of Neuroscience, 2nd Medical Faculty, Charles University, Prague.

     Studies at the Department are aimed at understanding the maintenance of ionic and volume homeostasis in the CNS, the extracellular space as a communication channel, the diffusion parameters of the extracellular space (ECS), extrasynaptic "volume" transmission and the role of glia in signal transmission, behavior and regeneration. To understand the changes that occur in pathological states, several animal models of pathological states and diseases attacking the CNS are used, e.g., models of chronic pain, ischemia and ischemic lesions, perinatal and early postnatal anoxia, brain edema, hydrocephalus, multiple sclerosis, Parkinson's disease, developmental disorders, tumors, changes during aging, Alzheimer's disease, and brain and spinal cord injury, as well as models of CNS damage evoked by chemical or physical factors such as neurotoxins and X irradiation. The research aims are the improvement of therapy for CNS diseases and the prevention of CNS damage.

     Research at the Department of Neuroscience focuses on the following main topics:
- The origin, mechanisms and pathophysiological significance of ionic changes in the extracellular space
- Membrane properties and function of glial cells
- Diffusion in the extracellular space: the underlying mechanism of extrasynaptic ("volume") transmission
- Extracellular space volume and geometry - factors affecting diffusion in the CNS in health and disease
- Studies using transgenic animals
- NMR studies

The origin, mechanisms and pathophysiological significance of ionic changes in the extracellular space

     Studies at the Department have revealed that the ECS is an important communication channel, whose ionic and chemical composition, size and geometry depend on neuronal activity and glial cell function. Ionselective microelectrodes (ISM) are used to measure the activity of biologically important ions in nervous tissue. Experiments employing K+, pH and Ca++ ISMs have revealed that transmembrane ionic fluxes during neuronal activity and pathological states result in transient changes in CNS extracellular space ionic com position. Glial cells play an important role in K+ and pH homeostasis, buffering any excessive rise in extracellular K+ and alkaline shifts resulting from neuronal activity.

Fig 1. Typical membrane current patterns of astrocytes,oligodendrocytes, glial precursor cells and neurons evoked by depolarizing and hyperpolarizing voltage steps in acute spinal cord slices.

     To better understand the role of glia in physiological and pathological states, the membrane properties of astrocytes and oligodendrocytes have been studied in acute brain and spinal cord slices using the whole-cell patch-clamp technique. In the gray matter of rat spinal cord slices, astrocytes, oligodendrocytes and their respective precursors can be distinguished based on their pattern of membrane currents and immunohistochemical identification. Astrocytes, oligodendrocytes and glial precursor cells in the spinal cord gray matter are also sensitive to glycine, GABA, glutamate, kainate and NMDA. These and other studies have demonstrated the presence of neurotransmitter receptors on glial cells.

     The cell membrane of astrocytes and oligodendrocytes is almost exclusively permeable for K+. Recent patch-clamp experiments in our laboratory have revealed a significantly higher concentration ofextracellular K+ around oligodendrocytes than around astrocytes. These and other results demonstrate that the increased accumulation of K+ in the vicinity of the oligodendrocyte membrane results from a smaller ECS volume around oligodendrocytes than around astrocytes. Heterogeneities in the nervous tissue, represented by "clusters" of compact ECS around oligodendrocytes, can facilitate K+ spatial buffering and selectively affect the diffusion of ions and other neuroactive substances in specific areas and directions.

Measuring glial membrane properties using the patch-clamp method (Miroslava Anderova).

     Although synaptic transmission is an important means of communication between neurons, neurons also communicate among themselves and with glia by extrasynaptic "volume" transmission, which is mediated by diffusion in the extracellular space (ECS). The ECS of the central nervous system (CNS) is the microenvironment of neurons and glial cells. The composition and size of the ECS change dynamically during neuronal activity as well as during pathological states. Following their release, a number of neuroactive substances, including ions, mediators, metabolites and neurotransmitters, diffuse via the ECS to targets distant from their release sites. Glial cells affect the composition and volume of the ECS and therefore also extracellular diffusion, particularly during development, aging and pathological states such as ischemia, injury, X.irradiation, gliosis, demyelination and often in grafted tissue. Recent studies also indicate that diffusion in the ECS is affected by ECS volume inhomogeneities, which are the result of a more compacted space in certain regions, e.g., in the vicinity of oligodendrocytes. Besides glial cells, the extracellular matrix also changes ECS geometry and forms diffusion barriers, which may also result in diffusion anisotropy. Diffusion therefore plays an important role in extrasynaptic transmission, for example in functions such as vigilance, sleep, depression, chronic pain, LTP, LTD, memory formation and other plastic changes in the CNS. ECS diffusion parameters affect neuron-glia communication, ionic homeostasis and the movement and/or accumulation of neuroactive substances in the brain.

     Diffusion in the ECS obeys Fick's law, subject to three important modifications. First, diffusion in the ECS is constrained by the restricted volume of the tissue available for diffusing particles, i.e. by the extracellular volume fraction (a). Second, the free diffusion coefficient, D, is reduced by the square of the tortuosity (l) to an apparent diffusion coefficient ADC = D/l², due to an increase in the path length for diffusion between two points and because the diffusing substance encounters membrane obstructions, glycoproteins, macromolecules of the extracellular matrix, charged molecules and glial cell processes. Third, the diffusion of substances may be affected by nonspecific uptake, k', a factor describing the loss of a substance across cell membranes. If we incorporate factors a, l and k' into Fick's laws, diffusion in the CNS is described fairly satisfactorily.

     Studies in our laboratories have shown that the extracellular volume fraction changes during development, being about twice as large in the cortex and corpus callosum of newborn rats as in adults. The large ECS in the developing CNS might allow for the more effective diffusion of macromolecules, such as growth factors and cytokines. The reduction in ECS volume fraction with increasing age correlates well with gliogenesis and myelination. Changes in the membrane currents of glial cells associated with myelination have also been correlated with ECS diffusion parameters.

     Extracellular space diffusion parameters were studied during aging. Aged rats were classified according to their performance during place learning, and two groups, good and bad learners, were selected. Diffusion measurements were performed along three orthogonal axes. The volume fraction and nonspecific uptake were significantly lower in both aged groups than in young adults. In young adults and good learners, anisotropy was found in the hippocampus; the anisotropy was lost in bad learners. The loss of anisotropy in the hippocampus of aged bad learners corresponded to the disorganization of glial processes and a loss of extracellular matrix (fibronectin and chondroitin sulfate proteoglycan). The significant differences in diffusion parameters between good and bad learners in the CA3 and DG regions of the hippocampus may affect LTP, memory and learning. During hypoxia, our experiments have revealed that a dramatically decreases, while l significantly increases. The time course of the changes is about ten times slower in neonatal rats than in adults, correlating with the wellknown resistance of the immature CNS to anoxia. These changes in diffusion parameters during and after ischemia enhance the accumulation of substances, contributing to brain damage and hindering the influx of metabolic substances during any subsequent reperfusion. Diffusion-weighted MRI studies have shown that the apparent diffusion coefficient of water is dramatically reduced during anoxia and ischemia, with a time course that correlates well with the observed changes in a and l. Further experiments are aimed at elucidating the mechanism underlying these changes in water diffusion during anoxia.

     Brain injury, with consequent neuronal death and astrogliosis, results in changes in CNS architecture. Changes in diffusion parameters in experimental models of injury and regeneration such as stab wounds and radiation necrosis have been compared with histopathological changes in order to elucidate their possible mechanisms. Experimental animal models have revealed that in the vicinity of the injury, ECS volume and the apparent diffusion coefficients of both water (ADC_w) and tetramethylammonium (ADC_TMA) are decreased due to cell death and astrogliosis. In the lateral region of the ipsilateral cortex, where no changes in ECS volume are found, prominent increases in extracellular matrix expression (chondroitin sulfate proteoglycan) are seen along with decreases of both ADC_w and ADC_TMA. This shows that the apparent diffusion coefficient of water is affected by diffusion barriers resulting from an increase in extracellular matrix.

     Following intracerebral bacterial inoculation, acute inflammation and increased blood-brain barrier permeability occurs, resulting in moderate changes in ECS diffusion parameters. More dramatic changes, particularly an increase in extracellular space volume, were seen in our studies utilizing an animal model of the demyelinating disease multiple sclerosis, experimental autoimmune encephalomyelitis, induced by an injection of myelin basic protein.

     Recent experiments in the Department have used the 6-OHDA-lesion rat model of Parkinson's disease to study two different grafting techniques and their influence on ECS diffusion parameters. Micrografting involves the transplantation of fetal dopaminergic cells into a number of small deposits in the striatum, while macrografting uses a single, larger deposit. We found a functional recovery, good survival of tyrosine hydroxylase-positive cells and astrogliosis in rats 3-5 months after grafting. Grafts were localized by T2 or diffusion-weighted NMR, and ECS diffusion parameters were investigated in the striatum. Tortuosity increased in the grafts and the adjacent tissue. The increase in ECS diffusion barriers corresponded to the astrogliosis in and around the grafts. The increased extracellular tortuosity therefore suggests an impediment to dopamine diffusion from the grafts into the lesioned striatum.

     Malignant brain tumors, particularly the gliomas, are among the deadliest of tumors; many patients, including children, die within 2 years. It has been suggested that the migration of cells is critically dependent on their shape and size, their binding to various proteins in the extracellular space (ECS) that can boost tumor invasiveness, and on the size and geometry of the ECS. The Department is currently studying the changes in ECS diffusion parameters in tumors and how these changes might affect drug delivery to brain tumors. Using diffusion analysis based on ion-selective microelectrodes and optical imaging, we have been studying the migration of molecules and drugs through the ECS in brain slices of normal and malignant tissue obtained from patients during neurosurgery. Attempts will be made to modify the diffusion parameters in order to decrease tumor invasiveness.

     Glial fibrillary acidic protein (GFAP) represents the major constituent of intermediate filaments and is specifically expressed in astrocytes. Increased GFAP expression is a characteristic feature of reactive gliosis. GFAP is necessary for the outgrowth of astrocytic processes in the presence of neurons. In view of the apparent importance of GFAP for astrocyte function, mutant "knock-out" mice lacking GFAP have been used in order to further study its function in vivo. In recent studies, our data have shown that GFAP-/- astrocytes swell less and more slowly than GFAP+/- astrocytes, suggesting that GFAP plays an important role in cell swelling during physiological as well as pathological states.

     To elucidate the influence of extracellular matrix components on diffusion in the extracellular space, we carry out experiments using mutant mice deficient in the expression of the glycoproteins tenascin-R and tenascin-C and HNK-1 sulfotransferase. Besides alterations in synaptic efficacy, a reduction of long-term potentiation and behavioral deficits, these animals also show dramatic changes in ECS diffusion parameters.

In other studies, transgenic animals are being used as a model of Alzheimer's disease. In these mice, a mutated APP23 gene results in an enhanced tendency for the animals to form amyloid plaques in their brains; these mice also show changes in ECS diffusion parameters.

     Presently, a great deal of attention is being paid to the research and clinical applications of MRI and MRS in biomedicine. In collaboration with the MR Unit of the Department of Diagnostic and Interventional Radiology at the Institute for Clinical and Experimental Medicine, we are investigating diffusion and perfusion in the brain and studying tissue metabolite concentrations by use of in vivo MR spectroscopy, utilizing an experimental spectrometer Bruker 4.7 Tesla.

     Part of our research involves the investigation of brain function in both humans and suitable animal models. The animal models focus on ischemic lesions, hypoxia, brain injury, hydrocephalus and transgenic animals. The MR data are compared with results obtained from diffusion analyses performed by the real-time iontophoretic method using ion-selective microelectrodes. The ultimate aim of this project is to make possible greater utilization and finer processing of MR spectra and MR images and to find a link between data obtained using MR imaging and changes in extracellular space diffusion parameters revealed by iontophoretic measurements.