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Research at the Department of Neuroscience focuses on three main topics:
I. Origin, mechanisms and pathophysiological significance of
ionic changes in the extracellular space.
II. Membrane properties, volume regulation and function of glial
cells.
III. Extracellular space volume and geometry - factors affecting diffusion
in the CNS.
Studies at the Neuroscience 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) and the role of glia in signal transmission and behaviour. To understand the changes which occur in pathological states, several animal models of pathological states and diseases attacking the CNS are used (e.g. chronic pain models, perinatal and early postnatal anoxia, ischemia and ictus, brain edema, multiple sclerosis, developmental disorders, tumors, behavioral changes during ageing and Alzheimer˘s disease) as well as models of CNS damage evoked by chemical or physical factors (e.g. neurotoxins, X?irradiation). The research aims are the improvement of therapy for CNS diseases and the prevention of CNS damage.
Electrophysiological methods established at the Neuroscience Department include intra- and extracellular recordings from neurones, intracellular recordings from glia, ion-selective microelectrodes, recordings of slow potentials, the real-time iontophoretic method to study diffusion parameters in nervous tissue, optical methods such as light transmission or light scattering, and the patch-clamp method. Standard morphological and immunohistochemical methods (e.g., GFAP, RIP, O1/04) are used to study morphological changes and extracellular matrix molecules and to classify glial cells. Experiments are performed on rats, mice and frogs, both in vivo and in vitro. In experiments in vitro, brain and spinal cord slices, isolated spinal cords and human tumor cells are used. In collaboration with other institutions, we use the method of nuclear magnetic resonance (NMR) and methods to test behavioral changes and learning deficits.
ORIGIN, MECHANISMS AND PATHOPHYSIOLOGICAL SIGNIFICANCE OF THE IONIC CHANGES IN THE EXTRACELLULAR SPACE
Recent studies at the Neuroscience 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. Ion-selective microelectrodes (ISM) were 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 (Fig. 1) and pathological states result in transient changes in CNS extracellular space ionic composition. 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. The numerous mechanisms by which glial cells control ionic homeostasis were studied.
Fig. 1. Increase in K+ activity in the extracellular
space surrounding a discharging neuron (action potentials), as recorded
by K+-selective microelectrode with K+- liquid ion-exchanger
at the tip.
MEMBRANE PROPERTIES, VOLUME REGULATION AND FUNCTION OF GLIAL CELLS
To better understand the role of glia in physiological and pathological states, the membrane properties of astrocytes and oligodendrocytes were studied in acute brain and spinal cord slices using the whole-cell patch-clamp technique. In the rat spinal cord, four types of glial cells, namely astrocytes, oligodendrocytes and two types of precursor cells, can be distinguished based on their membrane current pattern and distinct morphological features. The recorded cells were labelled and identified by immunocytochemical methods using cell-type-specific markers. The four populations of glial cells revealed distinct membrane currents, namely Na+, KDR, KA and KIR. We demonstrated that subpopulations of spinal glial cells respond to GABA, glycine, glutamate, kainate and NMDA. The glial populations, their currents and receptors undergo changes during postnatal development and in response to changes in the ECS volume and geometry.
EXTRACELLULAR SPACE VOLUME AND GEOMETRY - FACTORS AFFECTING DIFFUSION IN THE CNS
Although synaptic transmission is the major means of communication between nerve cells, it is not the only one. Substances can be released non-synaptically, diffuse through the ECS and bind to extrasynaptic, high-affinity binding sites. The neuroactive substances may diffuse through the ECS to target neurons, glia or capillaries without requiring synapses. This mode of communication can function between neurons as well as between neurons and glial cells, and may be a basis for the mechanism of information processing in functions involving large masses of cells such as vigilance, sleep, chronic pain, hunger, depression, plastic changes etc. On the other hand, impairment of ionic homeostasis and glial swelling during pathological states lead to compensatory shrinkage of the ECS, i.e. to dramatic changes in ECS architecture, which can contribute to the impairment of CNS function and neuronal damage.
Diffusion in the ECS obeys Fick's law, subject to two 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). The concentration of a released substance in the ECS is therefore greater than it would be in a free medium. 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 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 (Fig. 2). If we incorporate factors a, l and k' into Fick's laws, diffusion in the CNS is described fairly satisfactorily.
Changes in ECS diffusion parameters result from activity-related transmembrane ionic shifts and cell swelling under physiological conditions, e.g. electrical or adequate stimulation. In the spinal cord of the rat or frog, repetitive electrical stimulation results in an ECS volume decrease from about 0.24 to about 0.12, i.e. a decrease of as much as 50%. The changes in ECS diffusion parameters outlast the stimulation for many minutes or even hours. The diffusion parameters also differ during development and ageing. The ECS volume in cortex and subcortical white matter (corpus callosum) is almost doubled in the newborn rat (a = 0.30-0.40) and diminishes with age (a = 0.18-0.24), while the variations in tortuosity (1.5 - 1.6) are not statistically significant at any age. A reduction in ECS volume fraction correlates well with gliogenesis and myelination. We also provided evidence for anisotropic diffusion in the corpus callosum and hippocampus. Anisotropy may help to facilitate the diffusion of neurotransmitters (e.g. glutamate, GABA) and neuromodulators to regions occupied by their high affinity receptors located extrasynaptically. It may also be of importance for “cross-talk” between synapses, glutamate spillover, LTP, LTD and memory formation. Indeed, we found a loss of anisotropy in aged rats with a learning deficit.
Fig. 2. Scheme of the CNS architecture. CNS architecture is composed
of neurons, fibres, glial cells, cellular processes, molecules of the extracellular
matrix and pores between the cells. The architecture affects movement (diffusion)
of substances in the brain, which is critically dependent on pore size,
extracelluar space tortuosity, and cellular uptake.
Hypoxia, ischemia or terminal anoxia is accompanied by a decrease in the ECS volume in rat cortex or spinal cord from about 0.20 to about 0.04; tortuosity increases from 1.5 to about 2.2 and non-specific uptake significantly decreases. The same ultimate changes were found in neonatal and adult rats, in gray and white matter, in cortex, corpus callosum and in spinal cord; however, the time course in neonatal rats was about 10 times slower than in adults. In our recent studies using diffusion-weighted 1H MRS/MRI, we measured the apparent diffusion coefficient of water (ADCW). Anoxia evoked similar decreases in ADCTMA (measured by the iontophoretic method and ISMs) and ADCW (measured by the NMR method). Moreover, the time course of ADCW was the same as the decrease in ECS volume and tortuosity.
On the other hand, damage to the blood-brain-barrier, cell death, inflammation or edema formation, e.g. after X-irradiation or during EAE, resulted in an ECS volume increase and in acute phases, in a tortuosity decrease. However, in chronic lesions such as occur 1-2 weeks after X-irradiation, the volume fraction remains elevated and tortuosity increases, presumably partly due to astrogliosis. Indeed, in our recent experimental models of injury and in nervous tissue grafts, astrogliosis was accompanied by an increase in tortuosity and a persistant elevation of diffusion barriers.
The long-term changes in ECS diffusion parameters therefore affect: 1. Synaptic transmission (width of synaptic clefts, permeability of ionic channels, concentration of transmitters, dendritic length constant, etc.), 2. non-synaptic transmission by diffusion (diffusion of factors such as ions, NO, CO, transmitters, neuropeptides, neurohormones, growth factors and metabolites), 3. Neuron-glia communication, 4. ECS ionic and volume homeostasis. The long-term changes in local architecture affect tissue viability, the efficacy of signal transmission, plastic changes and changes in behaviour.
