Beyond the Neuron

Question 1

Brain cell classification flow diagram (10)

Answer 1

Excitable: Neurons

Non-excitable:
1) Non-Neuronal cells
-> Microglial

2) Neural Cells
-> CNS –> Macroglial (Astrocytes, oligodendrocytes, NG2 cells) + Ependymal cells

-> PNS –> Schwann cells + satellite cells + enteric glial cells

Question 2

Central Neuroglia + functions (3 + 6)

Answer 2

Astrocytes
Microglia
Oligodendrocytes

Glia:
- control the formation of synaptic circuits

-refine and remodel synapses and circuits

-can coordinate circuit ­wide neuronal differentiation

-regulate synapse formation and pruning

-adjust synaptic communication and plasticity

-regulation of ion homeostasis affects circuit function

Question 3

Astrocytes info (8)

Answer 3

Star-shaped and versatile

-Most numerous cell type in the brain - 30-50% of brain volume

-Guide the migration of developing neurons

-Act as K+ buffers and maintains homeostasis

-Involved in the formation of the BBB

-Function in nutrient transfer - >90% glutamate uptake

-Synaptogenesis and synaptic remodelling

Specialized Astrocytes:
Bergmann’s glial cell
Muller cell
pituicyte

Question 4

Oligodendrocytes info (5)

Answer 4

—-‘’few-branch’’ glia —–

• Myelination begins during fetal development, but proceeds most rapidly in infancy
• One oligo myelinates many CNS axons
• Target of autoimmune attack in MS

Specific oligodendrocyte myelin proteins:
PLP
DM20
MBP

Question 5

Microglia info (7)

Answer 5

• Microglia invade the brain shortly after birth= (forming “microglial fountains”)
• disseminates relatively evenly throughout brain parenchyma
  and acquire an idiosyncratic “resting” or “surveillant” phenotype
• Synaptic modification by microglial cells
• Microglia can phagocytose “weak” synapses => developing and adult-born neurons
• Microglial ILGF- 1 and BDNF = key mediators of synaptic plasticity
• The brain’s immune cell
• Release reactive oxygen products + inflammatory
  molecules triggered by DAMP’s

Question 6

What are the Embryonic germ layers involved following neurulation? (2)

Answer 6

Ectoderm: Astrocyte and oligodendrocytes

Mesoderm: Microglia and macrophages

Question 7

Why does Neurogenesis have an adaptive function in adults? (2)

Answer 7

Neurogenesis in adult brain has an adaptive function because newly produced neurons can integrate into and modify existing neuronal circuits

This continual supply of new neurons and glia then provides the postnatal and adult brain with an added capacity for cellular plasticity, although one that is restricted to a few specific zones within the brain

Question 8

Neural stem cell niche definition (2)

Answer 8

Defines a zone in which the stem cells are retained after embryonic development for the production of new cells of the nervous system

Critical to the maintenance of the stem cell niche are microenvironmental cues and cell-cell interactions that act to balance stem cell quiescence with proliferation and to direct neurogenesis versus gliogenesis lineage decisions

Question 9

Describe neurogenesis steps (4)

Answer 9

During development of the vertebrate CNS:
1) Neural tube

2) Neural stem cell niche

3) neurons are generated first

4) glial cells generated second -> astrocytes or oligodendrocytes

Question 10

What is repressed after neurogenesis? (2)

Answer 10

gliogenesis

via epigenetic silencing of genes that are necessary for astrocyte formation via DNA methylation and/or chromatin modifications.

Question 11

What does the neocortex of the adult brain consist of? (1)

Answer 11

neurons and glia that are generated by precursor cells of the embryonic ventricular zone.

Question 12

Radial glia facts (3)

Answer 12

• Radial glia are generated before neurogenesis and guide neuronal migration.
• Radial glia are mitotically active throughout neurogenesis, and disappear or become astrocytes when neuronal migration is complete.

-It has been suggested that radial glia may be neuronal precursors

Question 13

Cell generation steps (4)

Answer 13

1) Neuroepithelium
2) Neurogenesis
3) Gliogenesis
4) Postnatal

Question 14

Gliogenesis steps (6)

Answer 14

1) Neuroepithelial cells (neuroectodermal cells) form the wall of the closedneural tubein earlyembryonic development

2) Neuroepithelial cells proliferate and generate neuroblasts and immature neurons

3) They then differentiate into radial glia which proliferate and elongate

4) - Radial glia in the cortex contribute to neurogenesis directly orviaimmediate neuronal precursor cells
- Cortical and spinal cord radial glia contribute to gliogenesis by producing astrocytes and oligodendrocytes
- Some radial glia may also differentiate into ependymal cells which line the ventricles of the adult CNS

5) Towards the end of gliogenesis, many radial glia differentiate directly into mature astrocytes
- Subtypes of radial glia with distinct neurogenic and gliogenic potentials have been described, and radial glia-­derived progenitors persist through adulthood and give rise to adult neural stem cells

In addition to cells derived from the neuroepithelium, microglia, derived from erythro­myeloid­progenitors enter the vertebrate CNS early in development at the onset of neurogenesis, and subsequently proliferate and migrate to colonise the entire brain and spinal cord

Question 15

What do neuroepithelial progenitors do during the embryonic period? (3)

Answer 15

They take on characteristics of astrocytes

(including the expression of factors such as glutamate transporters, glycogen granules, and intermediate filaments)

These neuroeiptheial cell derived “radial glia” go on to generate neurons during embryonic stages of mammalian development, and then switch to generating mature glia (either directly by generating proliferating astrocytes, or through generation of intermediate progenitors, such as OPCs)

Question 16

What is the other source of immunocompetent cells in the brain? (1)

Answer 16

Macrophages infiltrating from the periphery

Question 17

Microgliogenesis (2)

Answer 17

The microglial population differentiates from the embryonic yolk sac, while peripheral macrophages are the monocytes originating from the hematopoietic stem cells and maturating in bone marrow.

Microglia regulate neuronal survival, phagocytose excess neurons undergoing apoptosis during both early development and in neurogenic regions of the adult brain, and have multiple roles in refining CNS formation and function

Question 18

Even though glia regulate neuron number - how can neurons regulate glial cell number? (2)

Answer 18

the necrosis of specific neurons influences initial microglial cell entry to the CNS + neuronal activity can regulate OPC proliferation and oligodendrocyte generation.

As will become clear, many aspects of nervous system formation and function are underpinned by dynamic bidirectional interactions of neurons and glia

Question 19

What are the main functions of glia? (4)

Answer 19

To surround neurons and hold them in place

To supply nutrients and oxygen to neurons

To insulate one neuron from another

To destroy and remove the carcasses of dead neurons (clean up)

Question 20

How do glia mediate synaptic networks? (4)

Answer 20

Key player in this is astrocytes - poorly understood how though

Propagation of intracellular calcium waves

Molecular mechanisms of astrocyte-induced synaptogenesis

Gliotransmitter release

Question 21

Whole-cell patch-clamp recordings from OPC (6)

Answer 21

measured their response to stimulation of afferent excitatory axons:

-OPCs were identified anatomically as smooth protoplasmic astrocytes based on their unique stellate morphology

-Glutamate receptor activation in these cells inhibits their proliferation and maturation into oligodendrocytes, and prolonged exposure to glutamate causes excitotoxic degeneration.

-Glutamate has been shown to reach other glial cells by diffusion from nearby synaptic clefts following vesicular release, or by reverse transport along axons.

This evidence of functional glutamatergic synapses between CA3 pyramidal neurons + CA1 OPCs in the hippocampus during the period of oligodendrocyte maturation, provides a pathway for axons to regulate myelination

Both electrophysiological and ultrastructural evidence shows that this synapse operates in the same manner as traditional neuron-neuron synapses. Transmitter at these synapses is released by vesicles, and the glutamate receptors activated are calcium-permeable AMPA receptors. It was the first neuro-glia synapse to be identified.

Question 22

Glia controls the formation of synaptic circuits (1)

Answer 22

Astrocytes in turn mediate synaptogenesis via both secreted [e.g.,hevin and secreted protein acidic and rich in cysteine (SPARC)] and membrane-bound factors (e.g., protocadherin and ephrin)

Question 23

Astrocyte-secreted factors induce synaptogenesis and specify circuit formation - 3 diagrams (4)

Answer 23

1) Astrocytes secrete thrombospondins (TSP) which bind to neuronal α2δ-1 to induce the formation of silent, structural synapses. The anti-epileptic drug Gabapentin (GBP) binds to α2δ-1, preventing TSP-induced synaptogenesis.

2) Astrocyte-secreted Hevin/SPARCL1 promotes synapse formation through its interactions with presynaptic NRX1α and postsynaptic NL1B, two proteins that do not normally interact. Astrocyte-secreted SPARC antagonizes Hevin-induced synapse formation through an unknown mechanism.

3) Astrocyte-secreted TGF-β1 promotes the formation of excitatory synapses through a mechanisms that requires NMDA receptor activity, along with the NMDA receptor agonist D-serine.

TGF-beta promotes formation of excitatory and inhibitory synapse formation

Question 24

Astrocytes can discriminate between neuron subtypes within a brain region and differentially modulating their synaptic activity - experiment (2)

Answer 24

a| In the mouse striatum, astrocytes selectively respond (by generating calcium signals) to the stimulation of distinct striatal neuron subtypes (specifically, those expressing either dopamine receptor D1 (DRD1) or DRD2) Stimulation of DRD1- or DRD2-expressing neurons results in the release of endocannabinoids that activate specific populations of astrocytes. In turn, astrocytes modulate synaptic activity selectively in homotypic pairs of neurons (expressing the same dopamine receptor) by releasing glutamate.

b| In the mouse visual cortex, photoactivation of astrocytes increases (in the figure, indicated by ‘+’) the spontaneous firing frequency of parvalbumin-expressing interneurons, whereas it either increases or decreases (in the figure, indicated by ‘+/−’) somatostatin-expressing interneuron activity. Whether astrocytes mediating these differential effects on interneuron subtypes are distinct subsets of astrocytes or are interchangeable is not known.

Question 25

What is neuronal excitability? (1)

Answer 25

A form of electrical excitability, determined by the existence of a specific complement of voltage gated ion channels in the plasmalemma Depolarization of the neuronal plasma membrane to a certain threshold activates these channels, which in turn generate regenerative action potentials that propagate mainly along the axon

Question 26

Glial cell excitability (4)

Answer 26

Mechanisms of excitability and signal propagation of neurones and glial cells are fundamentally different

Glial cells are electrically non-excitable and unable to generate action potentials.

Nevertheless, the glial cells are excitable, in the sense of responding to information from their surrounding.

The principal mechanisms used is Ca2+ signalling

Question 27

Astrocyte communication (4)

Answer 27

Stimulation of a single astrocyte evoked increases in [Ca2+]iin the simulated cell and in neighbouring astrocytes and Müller cells.

Glial Ca2+ signals are predominantly generated by Ca2+ release from the endoplasmic reticulum through opening of InsP3-gated Ca2+ channels

Endoplasmic reticulum membrane represents an excitable media that allows generation of propagating calcium waves, which integrate astroglial syncytia.

Neuroglial Ca2+ signals can be instrumental for integrative processes in neuronal-glial networks

Question 28

Astrocytes in vivo respond with global Ca2+elevations - experiment (4)

Answer 28

…During electrical stimulation of the locus coeruleus, during locomotion and during startle responses

Electrically stimulated LC neurons while monitoring Ca2+signals in cortical astrocytes.
They discovered global elevations in Ca2+upon LC stimulation, which is illustrated in the upper cartoons.
Similar global changes are seen when the mice are forced to walk or when they are startled.

Hence, during such responses, which may indicate a brain state change, the mode astrocyte Ca2+signaling shifts from random Ca2+microdomains and waves to global Ca2+elevations.

These global changes are mediated by noradrenaline release from LC projections, suggesting network wide alterations.

Question 29

Techniques to investigate precise intracellular machinery involved in the release of: (4)

Answer 29

Gliotransmitters

• glutamate
  -D-serine
• ATP

-Glial cells can produce and release neurotransmitter-like molecules, referred to as gliotransmitters, that can in turn influence the activity of neurons and other glia.

-One putative gliotransmitter, D-serine is believed to be an endogenous co-agonist for synaptic N-methyl-D-aspartate receptors (NMDARs), modulating synaptic transmission and plasticity mediated by this receptor

Question 30

Pathways mediating D-serine synthesis and release (4)

Answer 30

1)Activation of presynaptic neuron results in release of glutamate that binds to AMPA receptors on neighbouring astrocytes and causes release of D-serine

2) D-serine released from astrocytes binds to synaptic NMDAR-containing GluN2A subunits

3)Extra synaptic receptors
containing GluN2B preferentially
bind glycine instead of D-serine

4) L-serine is shuttled to neurons from
astrocytes through amino acid transporters (ASCT).

Question 31

What does D-serine do? (3)

Answer 31

-plays a particular important role in neurodevelopment

-Extrasynaptic receptors
containing GluN2B preferentially bind glycine instead of D-serine

-L-serine is shuttled to neurons from
astrocytes through
amino acid transporters (ASCT)

Question 32

How can we test if Individual astrocytes influence LTP induction mainly at nearby synapses? (1)

Answer 32

Clamping astrocytic Ca2+ blocks LTP at
nearby synapses in a
D-serine-dependent manner

Question 33

How do astrocytes respond to neurotransmitters? (4)

Answer 33

With calcium transients stimulating the release of gliotransmitters that regulate synaptic and neuronal functions.

Taken together, this suggests cortical astrocytes actively regulate the dynamic range of cortical neuronal network excitability that is responsible for sensory-evoked cortical gamma activity.

Combining in vivo two-photon astrocyte calcium imaging and electrophysiological recordings of neuronal network activity in the primary somatosensory cortex (S1),

cortical astrocytes exhibit stimulus-dependent and reliable responses to sensory stimulation. Calcium rises in astrocyte networks were found to be temporally associated with changes in neuronal network activity assessed by the spectral content of ECoG

Question 34

Microglia change synapse number via synaptic pruning (3)

Answer 34

Elimination of extraneous synapses (synaptic pruning) is critical for proper circuit function.

Synaptic pruning is regulated by neuronal activity and experience

Microglia dynamically interact with synapses and prune immature/inappropriate synapses

Question 35

Microglia + MHC1(2)

Answer 35

MHC class 1 proteins and complement cascade proteins have been suggested to contribute to the phagocytosis of synapses by microglia.

Activated microglia can also modify functional transmission at synapses via signalling pathways involving ATP, astrocytes, and glutamate receptors as indicated

Question 36

There has been growing evidence that BBB disruption is associated with … (2)

Answer 36

brain inflammatory conditions. These events would trigger the activation of microglial cells and promote localized damage to oligodendrocytes and the myelin sheath, ultimately compromising myelination and the integrity of neural circuits.

Question 37

What is the blood brain barrier and what is the neurovascular unit? (5)

Answer 37

Term recently coined to describe the interaction ofthe various cells inthe central nervous system withthe endothelial cell which makes up the blood brain barrier.

-The blood brain barrier is first a physical barrier.
-It’s a tight junctional membrane.
-It encompasses the inside of the capillary
-It is a transport barrier
-It controls the influx and efflux of transporters.

-It is also a metabolic barrier,these endothelial cells which surround the inside of the capillary.

-The neurovascular unit takes the endothelial cell andcouples it to surrounding astrocytes, neurons,and other cell types such as pericytes,microglia that are in the general area in the brain of the cerebral vascular capillary.

-Each of these cell types interacts with each other incontrolling the tight junction endothelial cell matrix.And it is a combination of these various cell types neurons, astrocytes, microglia,endothelial cells, pericytes that we term the neurovascular unit.

Question 38

What molecular triggers that lead to scar formation? (4)

Answer 38

-Epidermal growth factors (EGF)
-Fibroblast growth factor (FGF)
-Endothelin 1
-ATP

• Astrogliosis : Complex of molecular cascades which ultimately lead to circuit dismantling and subsequent reorganization of neural circuits

-Maintenance and formation of BBB

Question 39

How are surveillant and activated microglia activated? (3)

Answer 39

Surveillant microglia are activated by stimulating molecules through NF-κB signalling, upregulation of PU1 and CCAT binding.

Activated microglia and reactive astrocytes produce reactive oxygen species (ROS) and neurotoxic molecules (such as quinolinic acid) which can induce molecular processes leading to neuronal death.

Stimulatory molecules also induce reactive astrogliosis that leads to the upregulation of pro-inflammatory cytokine production, glutamate excitotoxicity and hyperexcitability of neurons