1

Diffusion in the extracellular space: the underlying mechanism of

extrasynaptic (volume) transmission

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.

Schematic of CNS
architecture, composed of
neurons (N), glial cells
(glia), neuronal and glial
processes (g), molecules
of the extracellular matrix
and intercellular channels
between the cells. This
architecture slows down
the movement (diffusion)
of substances in the brain,
which is critically
dependent on the ECS
diffusion parameters
volume fraction (a),
tortuosity (l) and
nonspecific uptake (k).

NEURON-GLIA COMMUNICATION VIA EXTRASYNAPTIC

TRANSMISSION

Eva Sykova
Dept. Neuroscience, Second Medical Faculty, Charles University and Inst. Experimental
Medicine ASCR, Videnska 1083, Prague 4, Czech Republic
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Extrasynaptic (volume) transmission plays an important role in short- and long-distance

communication between neurons, axons and glia [1,2]. It is mediated by the diffusion
of neuroactive substances through the extracellular space (ECS). In this way,
transmitters can reach high-affinity receptors located outside synapses and on glial
cells. The structure of cellular aggregates and the extracellular matrix (ECM) channel
the migration of molecules in the ECS, so that diffusion in certain regions is facilitated
in one direction rather than in another. This diffusion anisotropy has been found in
myelinated white matter, cerebellum and hippocampus [1,2] and may be of
importance for neuron-glia communication, 'cross-talk' between synapses, 'spillover' of
glutamate or GABA, LTP and LTD.

Changes in ECS volume and geometry accompany repetitive neuronal activity, seizures,
anoxia/ischemia, injury, gliosis, demyelination and many other pathological states. Ionic
changes and amino acid release related to activity, ischemia or CNS trauma result
either in fast and pulsatile or long-term cellular (particularly glial) swelling,
compensated for by ECS shrinkage and a decrease in the apparent diffusion coefficients
(ADC) of neuroactive substances or water as determined by diffusion analysis using ion-
selective microelectrodes, by diffusion-weighted NMR and by optical imaging. In
contrast, inflammation, oedema, cell loss and cell shrinkage during trauma and aging
result in an ECS volume increase. The concomitant structural changes, particularly
astrogliosis, result in an increase of diffusion barriers. A decrease in ADCs, i.e. an
increase in tortuosity, was found during astrogliosis or ageing in the rat cortex, corpus
callosum, hippocampus, spinal cord and in cortical grafts.

The movement of substances is hindered by the cell (particularly glia) swelling

narrowing the intercellular clefts and by diffusion barriers formed by changes in the
ECM and by the swelling of fine glial processes. Demyelination and gray and white
matter pathologies result in the loss of diffusion anisotropy. These changes can affect
the efficacy of neuron-glia communication, synaptic as well as extrasynaptic
transmission and induce damage to nerve cells by an increased accumulation of toxic
substances.

[1] E. Sykova, The Neuroscientist, 3:28-41, 1997.

[2] C. Nicholson and E. Sykova, Trends Neurosci, 21:207-215, 1998.

Supported by GACR 307/96/K226, 309/97/K048, 309/99/0657, VS96-130

ROLE OF GLIAL CELLS IN VOLUME TRANSMISSION

L. Vargova, E. Sykova
Dept. of Neuroscience, 2nd Medical Faculty, Charles University, and Institute of
Experimental Medicine AS CR, Videnska 1083, Prague, Czech Republic

Astrogliosis and cell swelling during neurological disorders and brain trauma may alter
the diffusion properties of nervous tissue [1]. Cell swelling and astrogliosis in vitro
were therefore evoked by the application of either 50 mM K+ or hypotonic solution (235
mmol kg-1) to isolated spinal cords of 4-21-day-old rats. As a model of reactive
astrogliosis in vivo, we used a cortical stab wound. ECS diffusion parameters - volume
fraction (a = ECS volume/total tissue volume), tortuosity l (l2= free/apparent diffusion
coefficient) and nonspecific uptake k' - were determined using the real-time
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iontophoretic method [2]. After the experiments, tissue sections were immunostained
for glial fibrillary acidic protein (GFAP) and chondroitin-sulphate proteoglycans (CSPG).

In spinal cord, both 50 mM K+ and hypotonic stress caused a decrease in a and an

increase in l. These changes were blocked in Cl--free solution and slowed down by
furosemide and bumetanide. After 10-20 min of K+ application, a started to return to
control values due to active regulation of cell volume (regulatory volume decrease -
RVD). RVD was blocked by the gliotoxin fluoroacetate. During washout in control
solution, a returned to and subsequently increased above control values by 50-100%
with a second rise in l. This rise in l correlated with an increase in GFAP staining and
astrogliosis. In white matter, where diffusion is anisotropic, 50 mM K+ and/or hypotonic
solution led to a greater increase of l along the fibres than across the fibres and to the
disappearance of diffusion anisotropy. Diffusion parameters in gliotic cortex were
measured 3, 7, 21 and 35 days post-wounding (dpw) in the hemisphere ipsilateral and
contralateral to the lesion. In the area 300-1000 mm around the wound, a was
increased at 3 and 7 dpw and returned to control values at 21 dpw; l was increased at
3, 7, 21 and 35 dpw, reaching a maximum at 7 dpw. Measurements made 1500-2000
mm from the wound revealed only an increase in l at 7 dpw. No changes in a and l were
found in the contralateral hemisphere. The time course of the changes correlated
closely with increased staining for GFAP and CSPG.

We conclude that acute swelling of glial processes as well as astrogliosis produce a

significant increase in diffusion barriers in the ECS and thus affect synaptic and
extrasynaptic transmission and neuron-glia communication.

Supported by GACR 307/96/K226, 309/99/0657 and VS 96-130.

[1] E. Sykova, The Neuroscientist, 3: 28-41, 1997.

[2] C. Nicholson and E. Sykova, TINS, 21: 207-215, 1998.

Quantitative Evaluation of 5-Hydroxytryptamine (Serotonin)

Neuronal Release and Uptake: An Investigation of Extrasynaptic
Transmission

Melissa A. Bunin and R. Mark Wightman

The Journal of Neuroscience, July 1, 1998, 18(13):4854-4860

Curriculum in Neurobiology and Department of Chemistry, University of North Carolina

at Chapel Hill, Chapel Hill, North Carolina 27599-3290

Whether neurotransmitters are restricted to the synaptic cleft (participating only in

hard-wired neurotransmission) or diffuse to remote receptor sites (participating in
what has been termed volume or paracrine transmission) depends on a number of
factors. These include (1) the location of release sites with respect to the receptors,
(2) the number of molecules released, (3) the diffusional rate away from the
release site, determined by both the geometry near the release site as well as binding
interactions, and (4) the removal of transmitter by the relevant transporter. Fast-scan
cyclic voltammetry allows for the detection of extrasynaptic concentrations of many
biogenic amines, permitting direct access to many of these parameters. In this study
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the hypothesis that 5-hydroxytryptamine (5-HT) transmission

is primarily extrasynaptic in the substantia nigra reticulata, a terminal region
with identified synaptic contacts, and the dorsal raphe nucleus, a somatodendritic
region with rare synaptic incidence, was tested in brain slices prepared from the rat.
Using carbon fiber microelectrodes, we found the concentration of 5-HT released per
stimulus pulse in both regions to be identical when elicited by single pulse stimulations
or trains at high frequency. 5-HT efflux elicited by a single stimulus pulse was
unaffected by uptake inhibition or receptor antagonism. Thus, synaptic efflux is not
restricted by binding to intrasynaptic receptors or transporters. The number of 5-HT
molecules released per terminal was estimated in the substantia nigra reticulata and
was considerably less than the number of 5-HT transporter and receptor sites,
reinforcing the hypothesis that these sites are extrasynaptic. Furthermore, the detected
extrasynaptic concentrations closely match the affinity for the predominant 5-HT
receptor in each region. Although they do not disprove the existence of classical
synaptic transmission, our results support the existence of paracrine
neurotransmission in both serotonergic regions.

Key words: 5-hydroxytryptamine; volume transmission; substantia nigra reticulata;

dorsal raphe; fast-scan cyclic voltammetry; transporter kinetics

Neuron-glia communication by volume transmission - quadrupartite

synapse

Eva Sykov
Institute of Experimental Medicine ASCR and Department of Neuroscience,
Charles University, Second Medical Faculty, Prague, Czech Republic

Synaptic transmission is an important means of communication in the central nervous

system (CNS); however, neurons themselves and neurons and glia also communicate by
extrasynaptic volume transmission, which is mediated by diffusion in the extracellular
space (ECS). The ECS of the 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 result in diffusion anisotropy. ECS size, geometry, and
composition, together with pre- and postsynaptic terminals and glial processes, form
the so-called quadrupartite synpase. ECS diffusion parameters affect neuron-glia
communication, ionic homeostasis and the movement and/or accumulation of
neuroactive substances in the brain. The ECS is therefore an important part of the CNS
and as such plays an important role in extrasynaptic transmission, transmitter spillover,
cross-talk between synapses, and in functions such as vigilance, sleep, depression,
chronic pain, LTP, LTD, memory formation and other plastic changes in the CNS.
 5

References:
NICHOLSON, C. and SYKOV, E. (1998) Extracellular space structure revealed by
diffusion analysis. Trends. Neurosci. 21: 207-215.
ZOLI, M., JANSSON, A., SYKOV, E., AGNATI, L.F. and FUXE, K. (1999) Volume
transmission in the CNS and its relevance for neuropsychopharmacology. Trends
Pharmacol. Sci. 20:142-150.
SYKOV, E. (2001) Glial diffusion barriers during aging and pathological states. Prog.
Brain Res. 132: 339-363
SYKOV, E. (2001) Glia and extracellular space diffusion parameters in the injured and
aging brain. In: Neuroglia in the Aging Brain, Ed. J. de Vellis, Humana Press, pp. 77-98
SYKOV, E. (2002) Plasticity of the extracellular space. In: The Neuronal
Microenvironment, Ed. W. Walz, Humana Press, pp. 57-81