Astrocytes marked with calcein-AM (green) in a cerebellar

granule cells culture, after oxygen-glucose deprivation.

Dr. Ana-Maria Zagrean Neuronal Cell Culture Lab

Interactiunea neuro-gliala

in modelarea

conectomului neuronal

Conf. univ. Ana-Maria Zagrean azagrean@gmail.com

• Sistem nervos (SN): totalitatea celulelor din SN si a

conexiunilor dintre ele - morfologice, functionale

(electrice/metabolice), chimice si fizice…

• SN: cea mai mare diversitate celulara,

• SN: cea mai mare diveristate a conexiuilor/tipurilor de

semnalizare intercelulare…

If ‘‘the connectome’’ represents a complete map of

anatomical and functional connectivity in the brain, it

should also include glia!

Neuron, Aprilie 2015

White Matter Connections Obtained with MRI Tractography From: Gigandet X, Hagmann P, Kurant M, Cammoun L, Meuli R, et al. (2008) Estimating the Confidence Level of White Matter Connections Obtained with MRI Tractography. PLoS ONE 3(12): e4006. doi:10.1371/journal.pone.0004006

Connectomics - Human Connectome Project

http://www.neuroscienceblueprint.nih.gov/connectome/index.htm

Charting neural connections is unlikely to deliver understanding perception,

consciousness, how the brain produces memories

BRAIN project risk to failure – it should consider that although the human brain

contains roughly 100 billion neurons, it contains billions more glia, non-electrical

brain cells, residing outside the neuronal 'connectome'.

Dismissed as connective tissue when they were first described in the mid-1800s, glia

have long been neglected in the quest to understand neuronal signalling.

‘Connectomica’ in sistemul nervos: mai mult

decat o problema de conexiune interneuronala….

20% din debitul cardiac Conexiuni interneuronale

Semnalizare electrica…

…glial trees have exactly the same level of detail and importance as neural trees,

yet they are ignored in the aspirations of the connectomists.

…grey matter might be imagined more like an astrocytic christmas tree farm

superimposed on a neural rainforest.

Risks of science… Myths in Neurosciences:

Past and Present!

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)

Past…

• Research is revealing that glia can sense neuronal activity and control it,

operate in diverse mental processes, as formation of memories.

• Glial cells have a central role in brain injury and disease, they are even at the

root of various disorders — such as schizophrenia and Alzheimer's —

previously presumed to be exclusively neuronal.

• Yet… the word 'glia' was not stated in any of the announcements of the

BRAIN Initiative, nor written anywhere in the 'white papers' published in

2012 and 2013 in prominent journals outlining the ambitious plan

• … urgent need for the community of neuroscientists behind the

initiative to expand its thinking!

Present…

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 – 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.

Astrocytes (red) and immature oligodendrocytes (green), types of glial

cell, intertwine with neurons (blue) from the brain's hippocampus. Nature 501, 25–27 (05 September 2013)

All major glial cell types in the brain - oligodendrocytes, microglia and astrocytes -

communicate with each other and with neurons by using chemical neurotransmitters

and gap junctions, channels that permit the direct transfer between cells of ions and

small molecules.

Neuropil - any area in the nervous system composed of mostly

unmyelinated axons, dendrites and glial cell processes that forms a

synaptically dense region containing a relatively low number of cell bodies.

Hippocampus

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

Oligodendrocytes (green)

• Form myelin electrical insulation, increasing conduction velocity by at least

50 times.

• Provide vital metabolic support for axons (purple).

Orthmann-Murphy, J. L. et al. J. Neurosci. 2007;27:13949-13957

Myelin, oligodendrocyts and network of intercellular channels

between astrocytes (A) and oligodendrocytes (O)

Microglia (green)

•Highly motile and responsive to nervous-system injury and infection.

•Monitor electrical activity in neurons and prune synaptic connections (red).

•Involved in almost all nervous-system diseases and in certain psychiatric conditions.

Microglia - 10-15% of all cells found within the brain

- the immune cells/resident macrophages of the brain and spinal cord (main

form of active immune defense in the central nervous

system, constantly scavenging the CNS for damaged neurons and

infectious agents and decreasing inflammation).

- release substances that stimulate repair.

- prune back synapses and rewire neural connections in a healthy brain

• ‘Synaptic pruning’ vs. apoptoza

• Pruning = indepartarea sinapsei inactive cu pastrarea

axonului si reorientarea catre alte sinapse, functionale

• Apoptoza=moarte celulara programata

• Scop:

-pastrarea preferentiala a sinapselor neuronale incluse

in retele functionale

-indepartarea neuronilor lezati/degenerati

-modelare functionala/plasticitate a sinapselor in scopul

imbunatatirii si/sau diversificarii functiei

Microglia

Astrocytes (red and green) influence nervous-system communication

and plasticity

•Primarily responsible for homeostasis of the central nervous system.

•Ensheath synapses, regulate neuronal excitability and synaptic

transmission.

•Respond to injury by secreting extracellular matrix proteins.

•Implicated in neurogenesis, cell migration, many neurological and

psychiatric disorders.

- Produce growth factors regulate morphology, proliferation, differentiation

or survival of neurons and glial cells

- 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 …

-Through perivascular processes astrocytes contribute to blood-brain barrier

and form "glymphatic" drainage system of the CNS.

-supplier of glutamine (neurotransmitters precursor).

-contribute to neuropathologies through mounting complex defensive

programme generally known as reactive astrogliosis.

Astocytes

Neurons alone provide only a partial explanation for complex cognitive processes,

formation of memories.

The complex branching structure of glial cells, integrating information from spatially

distinct parts of the brain and their relatively slow chemical (as opposed to

electrical) signalling in fact make them better suited than neurons to certain

cognitive processes.

Why are astrocytes important?

Different spatial and temporal

scales for neuronal and glial

activity…

Astrocytes marked with calcein-AM (green) in a cerebellar

granule cells culture, after oxygen-glucose deprivation.

Dr. Ana-Maria Zagrean Neuronal Cell Culture Lab

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%-20% 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

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

Physiological changes linking neural and vascular responses

http://www.scholarpedia.org/article/Neurovascular_coupling

Typical CBF

response to

brief neural

activation.

(CBF)

BBB & Neurovascular coupling

• The human brain contains on the order of 100 million

capillaries containing a surface area of 12 m2.

• Nearly every neuron in the brain has its own capillary, with

an average distance from capillary to neuron of 8–20 µm

Neurovascular coupling

The no. of astrocytes increases with an increase in

brain size:

The glia/neurons ratio

- in the rat cerebral cortex ~0.4,

- in the human cerebelar cortex ~1.65.

Increased complexity of astroglia in humans –

throught size & complex process arborization

Neuroglia in neurovascular coupling

one human protoplasmic astrocyte contacts and integrates ~2 million synapses

residing in its territorial domain, whereas rodent astrocytes cover ~20,000–120,000

synaptic contacts.

Astrocytes form a barrier around the blood vessels in the brain (astrocyte

end feet contributes to the blood-brain barrier formation and function).

Blood Brain Barrier functions

• BBB = structural and functional barrier which impedes and regulates the influx of most compounds from blood to brain

• BBB formed by – brain microvascular endothelial cells

– astrocyte end feet

– pericytes

• BBB is essential for normal function of CNS – Regulates passage of molecules in and out of brain to maintain

neural environment.

– Responsible for metabolic activities such as the metabolism of L-dopa to regulate its concentration in the brain.

Neuroglia in neurovascular coupling

- an important glial function - isolation of the nervous tissue from the rest

of the body by the blood–brain barrier (BBB)

- The barrier function of the cerebral endothlial cells is under astrocytic

control.

• 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

- secretion of soluble cytokines

- Ca2+ dependent signals triggered by intracellular IP-3 consecutive to ATP binding on P2Y receptors

- gap junction dependent pathways form a syncytium able to propagate signals (as Ca waves) for large distances

Astrocyte end feet

Blood brain barrier selectivity

• Free permeability (passive diffusion):

– small molecules: H2O, O2, CO2, NH3, ethanol

– lipid soluble molecules: steroid hormones

• Carrier mediated transport (apical-basal polarity):

– glucose: GLUT-1 (insulin independent)

– amino acids

– nucleosides, nucleobases

• Pinocytosis

• Aquaporin-4 is the main channel through which water

enters and leaves the CNS

• Physical blockage to paracellular diffusion (ions, peptides,

immune cells)

– astrocytes do not fire action potentials, but are Ca2+-excitable!

– astrocytes ‘listen’ to neurons (all major receptors present)

– astrocytes release neurotransmitter (Glutamate, ATP, …)

– astrocytes modulate neuronal excitability and synaptic transmission

Glial presence at synaptic level:

the tripartite synapse

Amzica, 2000

Synchronous Firing Groups:

Astrocytic regulation of neural networks

Neuron-glia connections

Synchronous Firing Groups – Astrocytic Regulation of Neural Networks

Neuro-glial connection – “calcium wave” in glial networks

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.

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, memory formation and

other plastic changes in the CNS.

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.

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)

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 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.

In loc de concluzii…

• Celulele gliale influenteaza activitatea si conectivitatea neuronala,

structural si functional; rol in transmiterea sinaptica si non-sinaptica…

• Eliberare de gliotransmitatori

• Prezenta receptorilor neuromediatorilor locali…

• “Sinapsa neuro-gliala”

• Specificitatea transmiterii semnalului

• Semnalizarea prin unde de calciu…

• Formarea de retele prin conexiuni gap (50-100 conexiuni gap cu alte

astrocite)

• Viteza de conducere a semnalului/informatiei…

• Mielinizarea este dependenta de activitatea neuronala

• Interactiunea neuro-gliala in formarea conexiunilor interneuronale, local

si la distanta; plasticitate neuro-gliala, neurogeneza si neurodegenerare

• Cuplarea neurometabolica si neurovasculara…

• Drenajul “glimfatic”

• Alterari la nivel glial in boli ale sistemului nervos

• ….

“Conectomul glial”, glie a conectomului neuronal…