WEEK 2

Question 1

neurocentrism

Answer 1

studies in the neurobiology of psychiatric and neurodevelopmental disorders have focused on the role of neurons, seen as the only determinants of behavior. there is now a shift to looking at glial cells as fundamental players in determining brain function, behavior, and mental health.

Question 2

silver impregnation

Answer 2

method to visualize astrocytes, developed by Golgi in 1873.

Question 3

Hann et al. 2013: do astrocytes influence behavior?

Answer 3

transplanted human glial progenitor cells in immunosuppressed mice. these progenitors gave rise to astrocytes with typical features of human ones.
FINDINGS: mice with human astrocytes performed better in cognitive tests and displayed improved LTP of synapses (plays a role in learning and memory).
CONCLUSION: astrocytes may be responsible for human cognitive abilities and can influence behavior.

Question 4

two ways that astrocytes influence behavior

Answer 4

1) directly: ability to release neurotransmitters (gliotransmission) and ability to form astrocytic networks

2) indirectly: involved in neuronal development and maintenance of a stable cellular environment - homeostatic role

Question 5

tripartite synapse

Answer 5

synapse composed of 3 elements: 2 neuronal elements (pre and post synaptic terminals belonging to two separate neurons) and an astrocytic process between them.

Question 6

gliotransmission

Answer 6

neurons release neurotransmitters and astrocytes respond to these neurotransmitters with elevations of calcium. then, in turn, they control neuronal excitability and synaptic transmission through calcium dependent release of gliotransmitters.
possible role in memory and sleep regulation. !!

Question 7

gliotransmitters

Answer 7

• glutamate
• GABA
• ATP
• adenosine
• d-serine

Question 8

Hann et al. 2012: astrocytes and memory

Answer 8

• impairment of spatial working memory in mice that lacked type 1 cannabinoid receptors on glutamatergic or GABAergic neurons. they also found impairment of LTD at hippocampal synapses in the glutamatergic and GABAergic neuron condition.
• preservation of spatial working memory in mice that lacked CB1R on astrocytes. preservation of hippocampal LTD as well.
  CONCLUSION: astrocytes play an active role in cognition and a role in its impairment in pathological state.

Question 9

astrocytes and sleep regulation

Answer 9

adenosine (sleep chemical) is released by astrocytes, and they play a role in regulating sleep homeostasis. astrocytes release ATP, which is converted to adenosine extracellularly. astrocytes can release gliotransmitters via exocytosis through the formation of a complex which is dependent on the SNARE protein.
in studies modifying mice so that the SNARE dependent release of gliotransmitters is abolished, both tonic and activity dependent extracellular accumulation of adenosine were prevented.
CONCLUSION: gliotransmission plays a role in the control of sleep.

Question 10

gap junctions

Answer 10

astrocytes can be directly coupled with neighboring astrocytes via gap junctions, which form aqueous channels between cells. these gap junctions allow the passage of ions and small molecules, therefore they allow direct intracellular communication. they are formed by 2 proteins: connexin 30 and connexin 43.
gap junctions also display selective permeability, which is age-specific and region-specific.

Question 11

calcium waves

Answer 11

coupling astrocytes in networks is the way in which this type of organization allows for calcium rises, generating calcium waves. spreading calcium waves may cause gliotransmitter release at remote synapses form the astrocytes that were initially activated.

Question 12

astrocytic networks and neuronal activity

Answer 12

2 kinds:

1) just an astrocyte and a synapse: neurotransmitters are released by the pre-synaptic neuron. these neurotransmitters act on receptors and transporters in the astrocyte. these will lead to the release of gliotransmitters, which in turn can influence neuronal activity.

2) glutamate that has been taken up by a neighboring astrocyte, and also glutamine, can diffuse and permeate through gap junctions of astrocytic networks. this trafficking may result in the subsequent release of gliotransmitters at the remote synapse or even at the extrasynaptic sites, affecting the activity of the underlying neuronal network.

Question 13

astrocytic networks: functions

Answer 13

1) regulate the generation of a rhythmic firing pattern in neurons - necessary for vital functions such as respiration and mastication.

2) function as a master hub, integrating the result of distributed processing from several brain areas and supporting conscious states.

Question 14

Pereira & Furlan (2010): astrocytic networks

Answer 14

proposed that astrocytic networks are essential for voluntary behavior. automatic behavior can only be executed by neuronal networks.
dysfunction of astrocytic networks could therefore lead to cognitive impairment.
it is not clear however if changes to the astrocytic network are a cause or a consequence of neuronal dysfunction!!

Question 15

why do we know so little about the potential role of astrocytes in psych disorder?

Answer 15

1) alterations may be subtle

2) people do not usually die of psych disorders

3) at the time of death, past history of disorder may be ignored

4) other illnesses may mask changes caused by the disorder

5) alterations caused by pharmacotherapy may be indistinguishable from changes caused by the disorder itself

Question 16

main lines of evidence

Answer 16

1) human studies: post-mortem

2) animal studies: use of genetically modified animals

3) in-vitro studies: astrocyte cultures, brain slices

Question 17

depression: hypotheses of underlying factors

Answer 17

1) the monoamine hypothesis: leading theory that posits that depression is the result of under activity of monoamine neurotransmitters, especially serotonin.

2) dysfunction of the hypothalamic pituitary adrenal axis (HPA axis), a system involved in the response to stress.

3) circadian rhythm abnormalities play a critical role in mood disorders.

4) neurodegenerative inflammatory alterations may contribute to especially late onset depression.

5) astrocytic atrophy?

Question 18

chronic unpredictable stress (CUS) in animals: astrocyte/depression study

Answer 18

animals were subjected for 35 days to the same sequence of 12 stressors, 2 per day. the authors measured the level of mRNA for a marker of astrocytes, GFAP.
FINDINGS: significant decrease in the level of GFAP mRNA in animals exposed to chronic stress. this effect was reversed by injecting the animal with a glutamate modulating drug, riluzole.

Question 19

sucrose preference test

Answer 19

used to measure anhedonia in mice. mice show a preference to sweet foods and solutions, even above water. reduced sucrose preference in a test where they are given the option of a sweet drink or water bottle represents anhedonia. a chronic antidepressant can reverse this reduced preference.

Question 20

CUS and anhedonia in rodents

Answer 20

day 15 of exposure to stress, rodents showed a significant decrease in sucrose preference (controls 3x more likely to drink from the sucrose bottle). this was even more significant for the rodents exposed to 35 days of stress. chronic riluzole treatment reversed this, in parallel with decreasing glial pathology.

Question 21

riluzole and pathophysiology of MDD

Answer 21

riluzole boosts glutamate uptake by astrocytes and glutamate production by astrocytes. patients with MDD and CUS rodents show low cortical levels of GABA. GABA synthesis in neurons requires glutamine, which is produced by astrocytes. SAUR the disregulation of astrocytic support of GABAergic transmission contributes to MDD pathophysiology.

Question 22

Torres-Platas et al.: decreased GFAP expression in depression suicides

Answer 22

measured levels of the astrocytic-specific marker GFAP in various brain areas of post-mortem material obtained from depressed suicides.
FINDINGS: in mood uninvolved areas, like the PVC and cerebellum, GFAP mRNA and protein levels were the same as controls. however, in mood involved areas, like the mediodorsal thalamus and caudate nucleus, both mRNA and protein levels were downregulated in depressed suicide individuals.

Question 23

fluoxetine and astrocytes

Answer 23

prozac works via serotonin uptake. however, it also can act on astrocytes.
study: astrocytes treated with prozac produced trophic factors. this effect is independent of serotonin: astrocytes treated with serotonin did not show this increase in trophic factors.
interestingly, the full therapeutic effect of prozac is delayed until 4-6 weeks, but if prozac only acted on serotonin the effect would be immediate. if the main effect is on astrocytes, however, the delay is better explained since it takes time for the increase in trophic factors to lead to the increase in uptake plasticity, neurogenesis, and restoration of damaged neuronal networks.

Question 24

Ernst et al.: disruption of astrocytic networks and MDD

Answer 24

gap junctions that create astrocytic networks are composed by connexin 30 and 43.
measured the levels of cx30 and cx43 in the PFC of suicides.
FINDINGS: both the levels of mRNA and proteins of cx30 and cx43 are reduced in the suicide cases.
CONCLUSION: link between dysfunction of astrocytic networks and depressed individuals.

Question 25

cx43 decrease in the PFC of stressed rats

Answer 25

both protein and mRNA of cx43 were significantly decreased in CUS rats. they were restored to normal levels through either of two antidepressants: fluoxetine or duloxetine.

Question 26

CUS disrupts astrocytic networks

Answer 26

Lucifer yellow was injected into the CUS rats’ brains. both the diffusion distance and the number of coupled astrocytes were reduced in the CUS rat group. these levels were restored to normal following antidepressant treatment.

Question 27

gap junctions and depressive-like behavior

Answer 27

authors infused the chemical carbenoxolone, which blocks gap junctions, into the PFC of rats. this induced depressive-like behavior, as shown by the sucrose preference test. in fact, even the lowest dose of carbenoxolone showed a decrease in preferential behavior.

Question 28

astrocytic networks and MDD: mechanism hypothesis

Answer 28

decreased expression of cx may alter calcium wave propagation and communication between astrocytes, possibly leading to a decrease in the simultaneous release of gliotransmitters and/or affecting the metabolic roles of astrocytic networks. therefore, astrocytic network dysfunction may play a role in depression.

Question 29

sleep deprivation, gliotransmitters, and MDD

Answer 29

astrocytes play a role in the regulation of sleep via SNARE dependent signaling mediated by the A1R neuron. sleep deprivation is a potent, short-term antidepressant with a 60-70% success rate. one or more nights of deprivation can alleviate sxs. this works on mouse models as well.
HOWEVER this reduction isn’t shown in A1R knockout mice, in mice that have reduced gliotransmitter release, and mice injected with an adenosine receptor antagonist. this suggests that antidepressive effects of sleep deprivation require gliotransmitter release from astrocytes, so there is an involvement of gliotransmitters in depression.

Question 30

radial glial cells

Answer 30

born from neuroepithelial cells, radial glial cells generate neurons in early development. later in development, radial glial cells generate astrocytes, oligodendrocytes, and adult neural stem cells, which are required to generate specific types of neurons in the adult brain.

Question 31

adult neural stem cells

Answer 31

generated by radial glial cells in later development. they are found in 2 locations in the adult brain:

1) the sub ventricular zones of the lateral ventricles

2) the sub granular zone of the dentate gyrus, which is part of the hippocampal formation

Question 32

neuroepithelial cells

Answer 32

the founder cells of the CNS. they have the ability to generate all the different cell types in the developing CNS, including all the different types of neurons and two types of glial cells, astrocytes and oligodendrocytes. this is why they are dubbed embryonic neural stem cells (NSCs).

Question 33

characteristics of embryonic NSCs

Answer 33

non-specialized cells that can:

1) self-renew

2) differentiate into appropriate specialized cells (neurons, astrocytes, or oligodendrocytes)

Question 34

self-renewal

Answer 34

ability of a cell to divide and generate two cells identical to the parent. it is needed to make sure cells don’t run out.

Question 35

differentiation: examples

Answer 35

• an embryonic NSC may divide and generate another embryonic NSC + a neuron
• an embryonic NSC may divide and generate a progenitor cell, like a radial glial cell + a neuron
• a radial glial cell can self-renew, dividing and generating another radial glial cell + a neuron
• a radial glial cell can divide and generate a dedicated progenitor cell (cell that can only generate a single cell type, like a neuron) + a neuron.

HOWEVER: radial glial cells cannot generate embryonic NSCs, dedicated progenitor cells cannot make radial glial cells or embryonic NSCs, and neurons cannot divide at all - they are terminally differentiated.

Question 36

asymmetrical differentiation

Answer 36

differentiation of a cell whereby the parent makes two different progeny.

Question 37

differentiation and specialization

Answer 37

embryonic NSCs are the least specialized cells, followed by radial glial cells, dedicated progenitor cells, and neurons.

Question 38

Cajal on adult neurogenesis

Answer 38

“once development ended, the fonts of growth and regeneration of the axons and dendrites dried up irrevocably. in the adult centers, the nerve paths are something fixed and immutable: everything may die, nothing may be regenerated” (1928)

Question 39

Altman & Das on adult neurogenesis

Answer 39

suggested with autoradiographic and histological evidence that some new neurons were indeed born in the adult hippocampus of rats, 1965. this effort to prove adult neurogenesis was later taken up in the early 90s.

Question 40

Frisen et al.: adult hippocampal neurogenesis

Answer 40

measured the concentration of C14 in genomic DNA in post-mortem human brains.
FINDINGS: there is C14 concentration in the hippocampal neurogenic DNA which occurs a time after the date of birth of the individual, demonstrating neurogenesis throughout life.

Question 41

adult neurogenesis: numbers

Answer 41

in the human adult hippocampus, we produce around 700 new neurons per day.

by the time we turn 50, we will have replaced the entire granular cell population we were born with with adult-born neurons.

in adult rodents: 70% of the bulbar neurons are replaced during a 6-week period.

Question 42

neuronal fate

Answer 42

determination of stem cells by extrinsic signals from the neurogenic environment.

Question 43

neurogenic environment: experiments

Answer 43

if you extract NSCs from a non-neurogenic environment, like the spinal cord, grow them, then replant them in a non-neurogenic environment, you do not get any neurons.

if you extract NSCs from a neurogenic environment, grow them, then replant them in a non-neurogenic environment, you still do not get any neurons.

if you extract NSCs from a non-neurogenic environment, grow them, then replant them in a neurogenic environment, like the dentate gyrus, you get neurons!

Question 44

what constitutes the neurogenic environment?

Answer 44

1) endothelial cells

2) proximity to blood vessels

3) astrocytes within the environment

Question 45

Song et al. (2002): neurogenesis, astrocytes, and adult NSCs

Answer 45

extracted NSCs from the adult hippocampus and co-cultured them with astrocytes - either extracted from the hippocampus or from a non-neurogenic environment (spinal cord).
FINDINGS: NSCs produced the most neurons when coupled with Neo-natal hippocampal astrocytes, followed by adult hippocampal astrocytes. spinal cord astrocytes produced lower numbers of neurons, comparable to control conditions without any astrocytes.

Question 46

Lie et al. (2004): molecular control of adult neurogenesis

Answer 46

adult NSCs express bone morphogenic protein, BMP, which instructs them to adopt a neural cell fate. however, in the neurogenic environment, the BMP inhibitor, noggin, is secreted by the ependymal cells and serves to block the gliogenic effect of BMP, driving the fate of the NSCs towards a neuronal fate.

Question 47

Nature (2005): WNT signaling and adult hippocampal neurogenesis

Answer 47

WNT signaling regulated adult hippocampal neurogenesis. they identified cells expressing WNT in the sub granular zone, where NSCs reside within the hippocampal niche.
they extracted adult hippocampal NSCs and cultured them in vitro with and without WNT factors.
FINDINGS: WNT pushed them towards neuronal fate, as indicated by the increased number of neuroblasts. there was in fact a 4-fold increase in neurons produced when NSCs were cultured in the presence of WNT.

they then injected the cells with a negative WNT antivirus blocking WNT signaling.
FINDINGS: the number of newborn neurons has decreased eightfold when WNT signaling was blocked.
CONCLUSION: WNT signaling is an important regulator of adult hippocampal neurogenesis.

Question 48

adult neurogenesis and learning + memory

Answer 48

the level of neurogenesis in the dentate gyrus is positively correlated with hippocampal-dependent learning tasks. if we block neurogenesis, we block hippocampal-dependent learning abilities. this is a bi-directional link: hippocampal-dependent learning can also modulate adult neurogenesis.

Question 49

neurogenesis and improved cognition: mechanisms

Answer 49

new neurons:
- increase memory capacity

• reduce interference between memories (pattern separation)
• add info about time to memories
• forgetting during infancy

Question 50

neurogenesis and depression

Answer 50

neurogenesis is reduced in animal models of depression, and many treatments for depression promote neurogenesis.
link: if you block neurogenesis in an animal model being treated with antidepressants, you will prevent the efficacy of the antidepressants, showing a link between neurogenesis and depressive behavior.

Question 51

modulators of adult hippocampal neurogenesis

Answer 51

• learning and social interaction increase neurogenesis. isolated rodents show a decrease in hippocampal neurogenesis
• stress (especially chronic stress) and chronic sleep deprivation decrease the level of hippocampal neurogenesis
• running increases neurogenesis
• aging decreases neurogenesis

Question 52

Villeda et al. (2011): aging and neurogenesis

Answer 52

• isochronic groups: young + young mice; old + old mice
• heterochronic groups: old mouse fused with young brain + young mouse fused with old brain

FINDINGS:
young+young = nice level of neurogenesis
young fused with old brain = decrease in the level of neurogenesis compared to the isochronic young group
old+old = dramatic decrease in the level of neurogenesis
old fused with young brain = increase in the level of neurogenesis compared to the isochronic old group

redid the experiment with plasma:
young mouse injected with young plasma showed a normal level of neuroblasts, whereas a young mouse injected with old plasma showed a dramatic decrease of neurogenesis.

Question 53

Praag et al. (1999): running and adult neurogenesis

Answer 53

neurogenesis can be influenced by an intervention such as running.
FINDINGS: an increase of nearly 30% of NSCs proliferating that will lead to neurogenesis in mice who ran compared to those who didn’t have access to a wheel.

Question 54

neurogenesis and diet

Answer 54

• limiting calorie intake by 30% or intermittent fasting increase neurogenesis
• flavonoids containing cocoa and fruit with dark skin increase neurogenesis
• omega-3 fatty acids in oily fish increase the production of new neurons
• diets rich in saturated fats decrease neurogenesis
• alcohol decreases neurogenesis, but reservatrol in red wine has a positive effect.
• soft food decreases neurogenesis
• all modulations also increase or decrease cognition and mood, suggesting that neurogenesis mediates the effect of diet on mental health!!

Question 55

pluripotent cells

Answer 55

cells that have the capacity to generate all the different cell types that make up both the fetal and adult body.
pluripotency is ephemeral: inner cell mass cells only have this property for a few days, then lose it, making it incredibly difficult to study.

Question 56

embryonic stem cells (ESCs)

Answer 56

population of stem cells derived directly from the inner cell mass, the culture of which retains pluripotent. compared to inner cell mass cells, ESCs derived from the inner cell mass have permanent pluripotency. just like the inner cell mass cells, ESCs can give rise to all cell types that make up the body.

Question 57

Gurdon: pluripotency in frogs

Answer 57

took enucleated eggs from frogs that are pluripotent, injected them with skin cell nuclei. the transplanted egg formed tad poles, meaning the cell remained pluripotent without its nucleus.
CONCLUSION: this suggests that there are factors in the cytoplasm of pluripotent cells that dictate pluripotency.

Question 58

Yamanaka factors

Answer 58

reduced a list of 24 factors that seemed to induce pluripotency to a definitive list of 4: Oct 3/4, Sox2, KlF4, c-Myc. If he left out those four there was no blue staining (activation of Fbx15, indicator of pluripotency) or there was some but a lot less efficient. just those four factors give rise to colonies. if he leaves any one out of the four out, it works much less efficiently, and any two factors don’t work at all.

Question 59

iPSC derivation regions

Answer 59

can be derived from:
- blood cells

• urine sample cells
• hair sample cells - useful for the study of childhood disorder as it is less invasive.

Question 60

Lancaster et al. (2013)

Answer 60

if you grow iPSCs appropriately, you can make them mini-brains, “cerebral organoids”, with real cortical structure. these cells have a capacity for histogenesis that is larger than any other neural developing system in vitro. eventually, however, the process stops as the brain can only get so large without blood supply.

Question 61

iPSCs and neurodevelopmental disorders: possible studies

Answer 61

1) we can take iPSC lines from an experimental group (with mental disorder) and a control group and compare if there are any differences in how patient lines grow

2) we can induce mutations in iPSCs via genome editing, as we know that many genetic variants contribute to different disorders.

3) we can study environmental risk factors: ASD is exacerbated by the mother having influenza in her first trimester, caused by cytokines. we can expose iPSCs to similar cytokines to see what difference it makes to their development

Question 62

iPSCs and neurodevelopmental disorders: possible phenotypes

Answer 62

1) we can look at gene expression: we can take lines from patients, or induce mutations, or expose the cells to environmental risk factors, and then study the differences in gene expression during development

2) we can look at physiology: these cells eventually become physiologically active, developing channels and receptors. we can look for electrophysiological properties that may be different during development

3) we can look at morphology: comparing the histologenesis of iPSCs derived from patient and control cells. do they form neurons in the same way? do their neurons have the appropriate structures?

Question 63

Pasca et al. (2011): Timothy syndrome iPSC comparisons

Answer 63

differentiated neurons from iPSCs derived from patients with Timothy syndrome and controls. Timothy syndrome is caused by a mutation of the CACNA1C gene which encodes a calcium channel - important for signal transduction in neurons.
FINDINGS: the Timothy derived iPSCs showed a phenotype that is exactly as you’d predict, with differences in the calcium channel mutation compared to controls.
the histogenesis was also the same: iPSCs derived from Timothy patients showed a greater propensity to make upper layer neurons and a weaker propensity to make lower layer neurons. within the lower layer neurons, there was a lower proportion of SATB2 positive cells. this was the exact same finding in transgenic Timothy syndrome mice, suggesting that the histogenic phenotype remains the same. the development of the cortex is altered in these cells.

Question 64

advantages of iPSCs

Answer 64

1) they are human cells

2) they have good construct validity (shown in the Timothy syndrome study)

3) we have good controls

4) tractable system

5) high through-put screening - good to discover novel drug targets

6) able to be manipulated genetically and phenotypically

Question 65

disadvantages of iPSCs

Answer 65

1) variability: no two individuals are the same, either genetically or epigenetically. at least mice can be genome controlled to become genetically identical

2) system properties are inaccessible: a disorder like autism or ADHD isn’t going to be a property of a single population of neurons, let alone a single set of molecules within a single population of neurons.

3) slow development: long wait (+/- 53 days)

4) no behavior: the cells don’t behave. we’d need additional clinical data and animal models to observe behavioral changes