Dottorato di Ricerca in Bioingegneria (XIII ciclo)
Politecnico di Milano
Modeling the electrophysiological properties of in vitroneurobiological systems:
communication in neuronal networks and collective electrical activity.
Michele Giugliano
I-7
Figure I.2: Levels of organization of the structure and function of the CNS: (from top to bottom)
level of molecules, level of individual cells, level of pairs of neurons connected by synapses, levl of
network of interacting neurons, level of systems in the brain that regulate behavior and level of
behavior (adapted from Levitan and Kaczmarek,1997).
Although the main focus of this thesis is on the modeling and simulation of cellular and molecular
phenomena, it must be emphasized that Cellular and Molecular Neurobiology do not exist in a
vacuum. Actually, most of the cellular organelles and sub-cellular mechanisms in the context of the
nervous system have their counterparts in other cell types, so that there is an emerging awareness of
a satisfying unity in Cell Biology. Moreover, it is becoming increasingly evident that the
understanding the nervous system requires a study at all the levels of organization depicted in figure
Dottorato di Ricerca in Bioingegneria (XIII ciclo)
Politecnico di Milano
Modeling the electrophysiological properties of in vitroneurobiological systems:
communication in neuronal networks and collective electrical activity.
Michele Giugliano
I-8
I.2, from behaving animls to single cells and to molecules that regulate cellular processes. As
already stated, no single level is inherently more important than any other, and for even a
rudimentary quantitative account of physiological and pathological phenomena in the CNS,
information and accurate experimental details from all the levels are necessary.
Since the mid of the nineteenth century, it was evident that discrete entities were the basic
architectural units constituting a living tissue. However, only fifty years later the same principle was
accepted in the context of the organization of the nervous system, as the passionate argument
between the two neuro-anatomists Santiago Ramón y Cajal ndCamillo Golgi, about whether the
brain consists of enormous numbers of discrete cells (i.e. the cellular hypothesis or neuron
doctrine), or is a continuous syncytium of tissue (i.e. the reticular theory), came to a successful end.
The answer to such a question was of enormous impact for understanding how signals spread from
one part of the nervous system to another. Thanks to a tissue staining technique, fortuitously
discovered by Golgi, Cajal managed to make individual neurons show up clearly in tissue sections
that actually contain a large number of neurons (see fig. I.3). In fact, by using other staining
methods that stain all the neurons, the same tissue sections would only have looked like tangl d
thickets under the optic microscope. Eventually, those discrete entities were correctly identified as
individual nerve cells, althoug Golgi never accepted such an interpretation and continued to put
forward his idea of a continuous meshwork. One of the reasons for the long debate over this issue is
the complexity of brain tissue: a huge number of different cell types composes the nerv us ti sues,
and many of these cells have a complex asymmetric three-dimensional morphologies that makes it
extremely difficult to ascertain where one cell ends and the next begins.
As it was already stressed, the essence of nervous system function and ofits ellular components is
signaling, both intra-cellularly (i.e. from one part of a cell to another) and inter-cellularly (i.e.
between cells). This is a fundamental premise for the following considerations, as most relevant
advances in the neurosciences came by investigating those specialized aspects of neuronal
morphology and structure contributing to the information transfer, to the mechanisms of intra-
cellular neuronal communications, to the patterns of neuronal connectivity and sub-cellular
mechanisms of intra-cellular signaling, to the relationship of various patterns of neuronal
connectivity to different behaviors, and finally to the ways in which neurons and their connections
can be modified by experience, in an activity-dependent fashion.
In particular, it can be further stated that each of the three unique sub-cellular str ctures
characterizing neuronal cells, being the axon (i. . specialized for intracellular information transfer),
Dottorato di Ricerca in Bioingegneria (XIII ciclo)
Politecnico di Milano
Modeling the electrophysiological properties of in vitroneurobiological systems:
communication in neuronal networks and collective electrical activity.
Michele Giugliano
I-9
the dendrites (i.e. the sites at which information is received from other neurons) and the synapses
(i.e. the points of information transfer between neurons) are highly devoted to signaling and
communication.
Figure I.3: Micrograph of a Golgi-stained single pyramidal neuron in the hippocampus: apical
dendrites (top), the soma and basal dendrites (middle - low) can be optically resolved through
staining (adapted from Levitan and Kaczmarek,1997).
The axon is a thin tube-like process, arising from the neuronal cell soma and traveling for distances
ranging from micrometers (e.g. in the retina) to meters (e.g. in the spinal-cord). Spec alized proteins
interleaved in the axonal membrane constitute the key mechanism allowing the neuron to rapidly
Dottorato di Ricerca in Bioingegneria (XIII ciclo)
Politecnico di Milano
Modeling the electrophysiological properties of in vitroneurobiological systems:
communication in neuronal networks and collective electrical activity.
Michele Giugliano
I-10
transmit electrical signals along the axon l ngth, from soma to the terminals (see the Methods). The
axon originates at a thickening on the cell body called the axon hillock, and it is often unbranched
until just before it terminates, where it may branch many times. Its diameter is approximately the
same throughout its length and structure, like the dendrite, and it is formed and maintained by the
cytoskeleton, a cellular scaffolding that is present in all cell types, however exhibiting unique
properties in unusually shaped cells such as neurons.
Dendrites are neuronal processes that tend to be thicker and much shorter compared to the axon,
and often highly branched, constituting a dense network of processes known as the dendritic tree
(see figure I.3). Moreover dendrites, whose cytoskeleton differs fr m that of axons, often originate
from the cell body, but in some neurons (e.g. in invertebrates) they arise even from the proximal
regions of the axon. Three-dim nsional computer reconstruction from images taken by means of a
confocal microscope, often reveal the presence of numerous finger-like small projections or
thickenings on the dendrites of some neurons. These projections, called dendritic spines, arise from
the main shaft of the dendrite (see fig. I.4) and on first approximation represent the synaptic input
sites at which the neuron receives information from another cell, although not necessarily all the
connections among neurons involve such structures.
Figure I.4: Three-dimensional computer reconstruction of a “spiny” dendrite from electron
scanning microscope sections.
Dottorato di Ricerca in Bioingegneria (XIII ciclo)
Politecnico di Milano
Modeling the electrophysiological properties of in vitroneurobiological systems:
communication in neuronal networks and collective electrical activity.
Michele Giugliano
I-11
Like the axonal membrane, the plasma membrane of dendrites contains specific proteins that allows
the dendrite to receive and integrate information from other nerve cells, by affecting the membrane
ionic permeability. The physiological role of the dendrite does not however consist exclusively in
gathering signals from other cells as, in a few cases, dendrites share with axons the ability to
actively transmit electrical signals (e.g. dendritic calcium spikes), and in many nerve cells both
input and output of electrical signals occur on the same set of dendrite-like fine proc sses.
The primary difference between neurons and most of other cell types (e.g. liver cells) is that
neurons can generate and transmit either electrical or chemical signals, being the messengers used
by the nervous system for all its functions. It is therefore of paramount importance to understand the
principles and mechanisms of neuronal signaling and communication. Despite the extraordinary
diversity and complexity of neuronal morphology and connectivity, a number of basic principles of
signaling for all neurons and synapses is adopted throughout the nervous system, collectively
referred to as excitability properties. In neurons or other excitable cells (e.g. muscle cells and
pancreatic β-cells), electrical signals are carried primarily by transmembrane ion currents, and result
in changes in transmembrane voltage. Four ion species are mainly involved in such currents:
sodium (Na+), potassium (K+), calcium (Ca2+) and chloride (Cl-), with the first three carrying
positive charges (i.e. cations) and the fourth carrying negative charges (i.e. anions). The flows of
these ions across the membrane are governed by physical laws and molecular mechanisms, later
reviewed, discussed and quantitatively modeled in the Method section of the present thesis, and
whose main energy source, ensuring ion movements, comes from ionic concentration gradients
between the cytoplasm and the extracellular environment. These gradients are indefinitely
maintained against thermodynamic equilibrium by active transport mechanisms called ion pump,
whose energy is derived from the hydrolysis of ATP molecules (i.e. adenosinetriphosphate). The
concentration gradients set up the electroch mical potential across the membrane, which drives ion
flow in accordance with the laws of diffusion and drift (i.e. at a first approximation on the basis of
the Ohm’s law). Although the energy sources and ion species involved in electrical signals are
relatively simple, the gating mechanisms modulating the passage of ions across the membrane and
determining the ionic membrane permeability, can be quite complicated. Actually, ions flow across
the membrane through aqueous pores formed by transmembrane protein molecules, also know as
the ion channels. These molecules may undergo three-dimensional conformational changes that,
under certain conditions, allow ion passage (i.e. gate in the openstate) but under other conditions
Dottorato di Ricerca in Bioingegneria (XIII ciclo)
Politecnico di Milano
Modeling the electrophysiological properties of in vitroneurobiological systems:
communication in neuronal networks and collective electrical activity.
Michele Giugliano
I-12
deny ion passage (i.e. gate in th closed state), depending on the transmembrane local electric field
as well as on the chemical interaction with ligand molecules. The quantitative description of ion
permeability and channel gating in biological membranes (i.e. both voltage-dependent and ligand-
gated protein channels) will be examined in the next chapters, because such phenomena are very
important for understanding the cellular bases of the electrophysiological collective behavior of a in
vivo as well as in vitro neuronal networks, represnting the cornerstones of neuronal signaling in the
nervous system.
I.2.2 Intercellular Communication
In the previous paragraph, it was outlined the relevance of the intercellular communication,
resulting in the information transfer from one part of the nerv us system to others, and being the
essence of nervous system function. It is just such a feature of an active information transfer that
distinguishes the nervous system from other organs. For such a reason, it is not surprising that the
neuron evolved a unique and highly specialized subcellular structure, the synapse, to carry out this
task (see fig. I.5).
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