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