Post on 08-Jun-2020
NeuroScience Optional Lecture
Physiology of the glial cells.
Neuronal microenvironement.
Ana-Maria Zagrean M.D., Ph.D
Cellular diversity of the brain
• Human brain ~100 billion neurons and several times as many
non-neuronal cells – the glial cells.
• Nervous system has a greater range of distinct cell types than
any other organ system, categorized by
– morphology,
– molecular identity,
– physiological activity
Glial cells
The prejudice that the relation between
neuroglial fibers and neuronal cells is
similar to the relation between connective
tissue and muscle or gland cells, that is, a
passive weft for merely filling and support
(and in the best case, a gangue for taking
nutritive juices), constitutes the main
obstacle that the researcher needs to
remove to get a rational concept about
the activity of the neuroglia.
S Ramon y Cajal
Nobel Price in Medicine 1906
Discoverer of the neuron (Blocks of brain soaked in silver nitrate)
Glial Cells – Non-neuronal Cells • Make up about 90% of the cells in the nervous system but 20%-
50% of the volume, depending on the nervous system region.
• Cannot generate or transmit nerve signals, but involved in
information processing.
• Responsible for the physical and metabolic support of the neurons,
but not only…
Types of Non-neuronal Cells
Four types associated with CNS:
-Astrocytes, oligodendrocytes,
microglia, and ependymal cells
Two types associated with the
peripheral nervous system
-Satellite cells and Schwann cells.
Glial Cell Functions:
Structural support, “glue”
Metabolic support (lactate shuttle)
Insulation (oligocytes)
Destroy pathogens, remove debris (microcytes)
In devolopment, guide axons
Release gliotransmitters (ex glut, ATP)
Regulate extracellular environment
Clear transmitters from synapse, ion homeostasis
K+ uptake vs. spatial buffering
Ependymal Cells
• Lines the ventricles or hollow spaces of the CNS
• Secretes cerebrospinal fluid
– Cerebrospinal fluid acts as a shock absorber and helps
to carry nutrients to the cells.
• Neurohormones transport
Microglia and Oligodendrocytes
• Microglia: phagocyte, remove pathogens and cell debris
from the brain; participate to the immune response.
• Oligodendrocytes wrap extension of their cell membranes
around sections of the axon (myelin sheaths).
– Myelin sheaths help to transmit nerve impulses.
Non-neuronal Cells of the Peripheral
Nervous System
• Satellite cells protect the cells of the ganglia
• Schwann cells/neurolemmocytes form a myelin
sheath around a segment of an axon. The myelin sheath
supports, protects, and insulates an axon.
A damaged axon can
regenerate, however, if at
least some neurilemma
remains.
Astocytes Star shaped with branching processes
Protoplasmic Astrocyte
Gray matter brain/spinal cord
Granular cytoplasm (cell body &processes)
Thick processes branch freely
Processes attach to blood vessels
(perivascular feet)
Smaller astrocytes = satellite cells
Fibrous Astrocyte
Between fiber tracts (white substance)
Fewer processes
Longer processes
Thinner processes (straighter/branch less)
Enwraps 4-8 neuronal somata and
300-600 dendrites.
Most prominent feature: Glial fibrillary
acidic protein (GFAP).
Gray matter - protoplasmic
White matter - fibrous
Confocal microscopy image of organotypic hypocampal brain-slice
stained for GFAP (red) to identify astrocytes and for MAP-2 (green) to
identify neurons.
Astocytes - particularities
Star-shaped cell that provides structural and nutritional
support - glucose/lactate; Metabolic support - lactate
shuttle
Also regulates K+ and neurotransmitters around synapses. Regulate extracellular environment.
The fine distal processes are interposed between all neuronal elements. Role in regulation of synaptic function. Neuron-glia connection. Network signalling
Processing information. Create a kind of synaptic island defined by its ensheathing processes.
Form a barrier around the blood vessels in the brain (blood-brain barrier). Keeps certain substances from moving into the brain.
Helps to direct neurons during embryo development and supplies the neurons with growth factors. Produce growth factors regulate morphology, proliferation, differentiation or survival of neurons and glial cells
Releases cytokines Glial cell conductances: Leak, Na-K pump, inward and
delayed rectifying K+ Release gliotransmitters (ex glut, ATP) Can also undergo remodeling (Plasticity) Receptors for hormones/neurohormones
More resistent to hypoxia/ischemia…
- Produce growth factors regulate morphology, proliferation,
differentiation or survival of neurons and glial cells - releases cytokines - Role in regulation of synaptic function. Volume transmission.
Neuron-glia connection. Network signalling - Can also undergo remodeling (Plasticity); astrogliosis in injury, neurodegeneration. - The fine distal processes are interposed between all neuronal elements. - Create a kind of synaptic island defined by its ensheathing processes.
-Processing information …
Astocytes
Premises for
neuron - glial cell - cerebral capillary unit
- Nervous system function ↔ cellular energetic status ↔
aerobic metabolism ↔ blood perfusion
- Brain vulnerability to hypoxia/ischemia
brain receives 15% from CO
O2 brain consume – 20% from the whole body
consume (250 ml O2/min)
glucose brain consume – 25% from the whole body
Brain Vulnerability
• Aerobic metabolism:
-95% of brain ATP derive from cerebral oxidative
phosphorilation
-No energy stores in the brain (low glycogen…)
• Facts - blockage of cerebral blood flow results in:
- loss of consciousness in 10-20 sec
- irreversible cerebral changes in 3-5 min
Oxidative vs. anaerobic metabolism
Non-oxidative (glycolysis)
TCA
Nucleus
mitochondrion
Oxidative (16 times more ATP)
glc glc
pyr
lac
Astrocyte-Neuron arrangement
– astrocytes do not fire action potentials, but are Ca2+-excitable!
– one astrocyte contacts 1000s of synapses!
– astrocytes listen to neurons (all major receptors present)
– astrocytes release neurotransmitter (Glu, ATP, …)
– astrocytes modulate neuronal excitability and synaptic transmission
Astrocyte end feet
• Star shaped glial cells
• Provides biochemical support for cerebral endothelial cells
• Influence of morphogenesis and organization of vessel wall
• Factors released by astrocytes involved in postnatal maturation of BBB
• Direct contact between endothelial cells and astrocytes necessary to generate BBB
• Co-regulate function by
the secretion of soluble cytokines
Ca2+ dependent signals by intracellular IP-3
gap junction dependent pathways
Astrocytes upregulate tight junction proteins, transporters (GLUT1…)
1. end-foot process (aquaporin4, K+ channel Kir4.1)
2. inducing factors (TGF-B, angiopoietins) recruiting periendothelial support
cells, impermeability of blood vessels
– astrocytes do not fire action potentials, but are Ca2+-excitable!
– one astrocyte contacts 1000s of synapses!
– astrocytes ‘listen’ to neurons (all major receptors present)
– astrocytes release neurotransmitter (Glu, ATP, …)
– astrocytes modulate neuronal excitability and synaptic transmission
Glial presence at synaptic level:
the tripartite synapse
Synaptic astrocytes 1. regulate synaptic transmission by - responding to ATP and glutamate, released
from the presynaptic neuron - uptake of glutamate from the synaptic cleft
via membrane transporters (green arrow) or the release of glutamate upon reversal of the transporter induced by [Na+]i
- D-serine released from astrocyte strengthen synaptic transmission by coactivating NMDA receptors in the postsynaptic membrane, or reduce synaptic transmission by secreting transmitter-binding proteins (TBP)
2. communicate with adjacent astrocytes via gap junctions and with distant astrocytes via extracellular ATP.
3. the rise in Ca2+ causes release of glutamate from astrocytes, and ATP is released via an unknown mechanism, which propagates ATP signaling to adjacent cells.
GluR, glutamate receptor; Ado, adenosine; IP3,
inositol trisphosphate; P1, adenosine receptor; P2, ATP receptor.
An electron micrograph of a synapse surrounded
by an astrocyte (yellow) from rat spinal cord.
Amzica, 2000
Modulation and optimization of synapse:
Regulation of chemical synapses function by
neuron – astroglia connections
GLAST = glutamat/aspartate transporter;
GLT-1 = glutamate transporter EEAT2
both Na+-dependent
[glutamate]i < 2-5 µM
[glutamate]o < 1-10 mM
Ca2+
Ca2+
- Evoked vesicle release
∝ presynaptic Ca2+ Astrocyte
Glu
Ca2+ - Neurotransmitter resource
limited --> depletion of vesicles,
refractory times, postsynaptic
depression
- Activity dependent increase in
presynaptic Ca2+ and synaptic
transmission probability
Modulation and optimization of synapse
Optimal synaptic potentiation
by astrocyte
When glucose phosphorylation is limited by the low brain glucose concentration,
astrocytic glycogenolysis can provide the necessary glucosyl units to maintain ATP
synthesis in the glial compartment (black lines).
Glycogen can provide fuel to neurons, presumably in the form of lactate, during
hypoglycemia and thus reduce the energy deficit in the neuronal compartment. When
the glucose supply is sufficient, glucose is stored in glial glycogen (blue lines).
Glc, glucose; Glc-6-P, glucose-6-phosphate; Lac, lactate.
Proportion of energy used by
glial cells is estimated from
14-17.5% to 30-40% of brain
oxidative metabolism.
Scheme of compartmentalized glial glycogen metabolism.
The Magistretti Hypothesis
• Astrocytes anaerobically metabolize glucose to lactate
• Neurons aerobically metabolize lactate/pyruvate
Magistretti (2000) Brain Research 886:108
Physiological coupling of brain metabolism and neuronal activity:
Glutamate-induced glycolysis in astrocytes
phosphoglucokinase
As Neural activity there is an Energy requirement
To solve this…
Astrocytic uptake of Glutamate leads to> ADP leads to> Glycolysis within Astrocytic endfeet which finally leads to > Lactate delivered to neuron
Ca2+
Ca2+
Evoked vesicle release
∝ presynaptic Ca2+
Astrocyte
Glu
Ca2+
Neurotransmitter resource limited
--> depletion of vesicles,
refractory times, postsynaptic
depression
Activity dependent increase in
presynaptic Ca2+ and synaptic
transmission probability
Modulation and optimization of synapse
Optimal synaptic potentiation
by astrocyte
Gliotransmission
Glutamate:
Post synaptic - contribute to network synchronization Pre synaptic - facilitates subsequent glutamate release. Favoring neurotransmission - ionotropic receptors Inhibition - metabotropic receptors
Ca2+
Astrocyte
Glu
Ca2+
Neuron to Astrocyte Signaling
1. Glutamate release from
pre-synaptic neuron
2. Metabotropic receptors for
Glutamate (mGluR) located
on astrocyte bind synaptic
Glutamate. Subsequent
intracellular Phospholipase C
release leads to Inositol
Triphosphate (InsP3)
production.
3. Ion channels open,
allowing vesicular-
bound pools of Ca2+
into the intracellular
environment.
4. Intracellular levels
of Ca2+ rise., free
Ca2+ releases other
pools of vesicular-
bound Ca2+.
- Regulation of ion concentration in the ECS:
Ex: High number of K+ channels (high permeability).
Transfere of K+ to sites of lower accumulation. High levels of K+ in ECS would
change neuronal exitability.
- Clear neurotransmitters (glutamate and GABA):
Astrocytes have distal processes rich in transporters that remove excess
neurotransmitters (especially glutamate)
If Glutamate is not removed:
Diffuses into the ECS. Presynaptic bind and inhibition of its own release.
Influence other synapses - “Intersynaptic cross-talk”
- Secrete large complex substances to the ECS: Important as structural
elements and cell to cell communication.
Ex: Promotion of the myelinating activity of oligodendrocytes through release of
cytokine leukemia inhibitory factor (LIF).
- Nervous system repair: upon injury to nerve cells within the central nervous
system, astrocytes become phagocytic to ingest the injured nerve cells. The
astrocytes then fill up the space to form a glial scar, repairing the area and
replacing the CNS cells that cannot regenerate
Astrocyte Role
-Vasomodulation: Restrict access of neurosecretory terminals to perivascular basal lamina. (blood flow) Control the effect of paracrine/autocrine secreted peptides. Regulate neurosecretion.
- Modulation of synaptic transmission
Neuron to Astrocyte Signaling
I. Glutamate release from
pre-synaptic neuron
II. Metabotropic receptors
for Glutamate (mGluR)
located on astrocyte bind
synaptic Glutamate.
Subsequent intracellular
Phospholipase C release
leads to Inositol
Triphosphate (InsP3)
production.
III. Ion channels
open, allowing
vesicular-bound
pools of Ca2+ into
the intracellular
environment.
IV. Intracellular levels
of Ca2+ rise., free
Ca2+ releases other
pools of vesicular-
bound Ca2+.
Gliotransmission
Glutamate:
Post synaptic - contribute to network synchronization Pre synaptic - facilitates subsequent glutamate release. Favoring neurotransmission - ionotropic receptors Inhibition - metabotropic receptors
Ca2+
Astrocyte
Glu
Ca2+
Amzica, 2000
Synchronous Firing Groups:
Astrocytic regulation of neural networks
Neuron-glia connections
Synchronous Firing Groups –
Astrocytic Regulation of Neural Networks
Calcium imaging reveals communication between neurons and glia.
(A) Molecules released during synaptic transmission bind receptors on glia that cause
increases in intracellular Ca2+ (rainbow colored cells), which are propagated as waves
through glial networks.
(B) Increases or decreases in axonal firing may coincide with the passage of a glial Ca2+
wave. Oligodendrocytes (purple) myelinate CNS axons. Vm, membrane voltage.
Neuro-glial connection – “calcium wave” in glial networks
From Fields and Stevens-Graham, 2005
Astrocytic Mobility
- Constantly changing their morphology. - Specially distal processes devoid of GFAP are extremely mobile. (GFAP imunolabelings show even in normal conditions)
Long term potentiation (LTP) Observed in Hippocampus - increase of density and closer
apposition to synaptic cleft of potentiate synapses.
Astrocyte Remodeling.
Examples:
Neuron-glia communication by volume transmission - quadrupartite synapse Neurons-to-neurons and neurons to glia communication by extrasynaptic “volume” transmission,
which is mediated by diffusion in the extracellular space (ECS) of the CNS = the microenvironment
of neurons and glial cells.
Composition & size of ECS change dynamically during neuronal activity and 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 also extracellular diffusion, particularly
during development, aging & pathological states (ischemia, injury, gliosis, demyelination).
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 plays an important role in
extrasynaptic transmission, transmitter spillover, cross-talk between synapses, and in vigilance,
sleep, depression, chronic pain, LTP, LTD, memory formation and other plastic changes in the CNS.
Basic concepts of ECS. A. Electronmicrograph of small region of rat cortex
with dendritic spine and synapse. The ECS is
outlined in red; it has a well-connected foam-
like structure formed from the interstices of
simple convex cell surfaces. Even though the
ECS is probably reduced in width due to fixation
procedure it is still evident that it is not
completely uniform in width. Calibration bar
approximately 1 μm.
B1-B4. Molecules executing random walks reveal
ECS structure.
C. Factors affecting the diffusion of a molecule in
the ECS. These are: a) geometry of ECS which
imposes an additional delay on a diffusing
molecule compared to a free medium. b) dead-
space microdomain where molecules lose time
exploring a dead-end. Such a microdomain may
be in the form of a ‘pocket’ as shown but it may
also take the form of glial wrapping or even a
local enlargement of the ECS. c) Obstruction in
the form of extracellular matrix molecules such
as hyaluronan. d) binding sites for the diffusing
molecule either on cell membranes or
extracellular matrix. e) fixed negative charges,
also on the extracellular matrix, that may affect
the diffusion of charged molecules.
• The ECS occupies a volume fraction of between 15 and 30% in normal adult brain
tissue with a typical value of 20% and that this falls to 5% during global ischemia.
• Volume fraction is denoted by α and may be formally defined as
where the subscripts on V denote the respective volume of ECS or the whole tissue
measured in a small region of brain (sometimes referred to as a Representative
Elementary Volume) and written as a decimal i.e. 0.15 ≤ α ≤ 0.3 .
In a ‘free’ medium, such as an aqueous solution or very dilute gel, α = 1.
• A recent study using quantum dot nanocrystals indicates that the true average width
of the ECS in the in vivo rat cortex lies between 38-64 nm.
• Relation with osmotic pressure, state of activity (e.g. glutamate release, [K+]o
increase), cell swelling…
Reduction in extracellular space and impaired diffusion thus may contribute to the
greater local accumulation of neuroactive substances and facilitate the development
of epileptic seizures.
Immature brains, with larger ECS, might reduce seizure susceptibility…
• Glial cells play an important role in modulating ECS diffusion parameters (e.g.
swelling of astrocytes, possibly in response to the stimulus-induced rise in [K+]o)
Diffusion in the extracellular space (ECS)
Extracellular microenvironment and volume transmission. Synapses and the entire ECS are embedded in an extracellular matrix of unknown density. The extracellular matrix has several components, including lecticans, tenascin-R (TN-R), as well as tenascin-C in the developing brain, and hyaluronan (HA). G, glia; N, neuron.
Extracellular communication. Short distance communication occurs via closed synapses that are typical of synaptic transmission and are often ensheathed by glial processes and by the extracellular matrix, forming perineuronal or perisynaptic nets. The ECS changes its diffusion parameters in response to neuronal activity and glial cell rearrange-ment. Presynaptic terminals, postsynaptic terminals, glial cell processes and the ECS form a ‘plastic’ quadripartite synapse. Long-distance communication. CNS architecture is composed of neurons, axons, glia, cellular processes, molecules of the extracellular matrix and intercellular channels between the cells. This architecture slows down the movement (diffusion) of substances in the brain. From Syková E, 2008
Retraction of glial processes in rat supraoptic nucleus (SON) and consequences for diffusion and synaptic crosstalk. Reduced astrocytic coverage of SON neurons in lactating rats leads to deficient glutamate clearance, resulting in increased glutamate concentration in the ECS, increased crosstalk between synapses and increased activation of either presynaptic or postsynaptic receptors.
Extracellular microenvironment
• diffusion studies were undertaken to determine the
critical mass of agent required to generate a seizure.
• agents that either cause seizure activity (penicillin) or
suppress it (valproate and verapamil) when introduced
into the cortex.
• Penicillin and pentylenetrazol are important in generating
seizure models for drug tests and other purposes
Astrocytes are connected by gap
junctions thereby forming a
syncytium that is able to propagate
signals for large distances
- Can be also caused by increased extracellular K+ levels.
- Modify gene expression and consequent morphological changes. - Cause own release of glutamate. Further adjacent neuron activation (not confirmed)
Ca2+ increase…
Matainance of microvascular tone
- Wave propagation signal - Mechanism of wave propagation via release of ATP to ECS > Activates neighboring cells. - Thigh junctions. Not certain. Observed only in intense electrical stimulation
Ca2+ Increase cause…
ATP and adenosine: ATP - P2Y receptors in astrocytes. Triggers intracellular Ca2+ release and wave propagation. > Glutamate Signal neighboring neurons by pre/post synaptic purinergic receptors. Converted to adenosine by ectonucleotidases in ECS. Suppression of synaptic transmission. A1/A2 receptors activation leads to positive action of K+ channels and negative action of Ca2+ channels.
Neuropathological conditions
Epilepsy - acompained by astrocyte hypertrophy and hyperplasia -Elevated baseline [K+]o has been directly correlated with the likelihood of
transition from interictal to ictal epileptiform activity (Jensen et al. 1994)
-Pharmacological block of K+ influx through K+ channels into glia causes an
abnormal accumulation of K+ in the extracellular space and an increase in
neuronal excitability (Ballanyi et al. 1987; D'Ambrosio et al. 1998; Gabriel
et al. 1998)
Astrocytes activated by injury - regulation of synaptic activity and strength. Importance in development of inflammatory pain.
Any structural change in astrocyte environment should affect properties of ECS.
The role of astrocytes in Epilepsy
– In astrocytes from epileptic foci
mGluRs are overexpressed by a
factor of about 20 (rat models and
human) Ulas et al., Glia 30, 352 (2000), Tang and Lee, J.
Neurocytology, 30, 137 (2001),
Aronica et al. Europ.J. Neurosci., 12, 2333 (2000)
– Enhanced IP3 Hydrolysis and
increased Ca2+ spikes during epileptic
seizure
Ong et al. J. Neurochem. 72, 1574 (1999)
– More spontaneous astrocytic
calcium spikes in epileptic foci
Tashiro et al., J. Neurobiol. 50, 45 (2002)
Higher abundance of mGluRs
Ca2+
Ca2+Ca2+
Ca2+ Ca2+Ca2+
Ca2+
Ca2+
Ca2+Ca2+
IP3
ATP, Glutamate
uptakeCa2+
leak
Endoplasmic
Reticulum
Abnormalities in glial biology contribute
to the pathology of schizophrenia.
• Neuregulins (NRG) are required for initial differentiation
of oligodendrocyte precursors and for their survival.
• A variant of this idea is that a deficiency of glial growth
factors––such as NRG––predisposes to synaptic
destabilization.
• It is clear that NRG signaling is required for the
stabilization of nerve-muscle synapses, and evidence for
NRG involvement in astrocyte biology might implicate
neuregulins in formation or stabilization of central
synapses.
Imaging brain cells metabolism
• Astrocytes (red cells) and neurons (blue cells) were labeled with specific antibodies in this fixed rat brain section. Because NADH, the coenzyme involved in brain metabolism, fluoresces differently in astrocytes and neurons in living brain tissue, biophysicists at Cornell could determine precisely when astrocytes were providing extra lactate "fuel" to neurons, confirming the controversial astrocyte-neuron lactate shuttle hypothesis
(K. Kasischke, P. Fisher/Cornell)
http://www.news.cornell.edu/AAAS96 /cellphoto.html
Astrocytes marked with calcein-AM (green) in a cerebellar
granule cells culture, after oxygen-glucose deprivation.
Dr. Ana-Maria Zagrean Neuronal Cell Culture Lab
From Selmeczy Z, 2008: Role of nonsynaptic communication in regulating the immune response
Nonsynaptic communication in the central nervous system