Week 2 Topic 1 - From embryonic NPCs to AHN

Question 1

What are the consequences of the gliotransmission - that is the release of gliotransmitter by astrocytes?

And what are the consequences of astrocytic modulation at the tripartite synapse for brain function and behaviour?

The simple answer is, we don’t know for sure. But there is some
evidence for a possible role in memory and in sleep regulation.

Answer 1

For what concerns their role in memory, evidence include the finding that, in brains slices, astrocyte activity can modulate long term potentiation LTP - as I mentioned before, a strengthening of synaptic connectivity, a mechanism, which is thought to underlie memory information as you have seen in the reading that you have just performed.

Other experiments in vivo also support the idea of an involvement of astrocytes in cognition.

Question 2

Before moving on to discuss the evidence for a contributing role of astrocytes to mental health
disorders, we should attempt to define what these disorders are.

Answer 2

In part one, we’ve seen how astrocytes, arguably, play an active information processing role in the CNS further to their supporting homeostatic role.

Therefore, it is logical to conclude that astrocytic dysfunction may well contribute to the development of mental health disorders.

Furthermore, we could argue that the lack of universally effective pharmacological treatments, or other form of treatment, for mental health disorder is due to the currently predominant ‘neurocentric’ approach to the study of human behaviour or mental illness.

This neurocentric approach has not allowed us to gain a full understanding of the mental illness.

Therefore, we could also argue that an alternative, gliocentric view, may lead to a better understanding of the basics of mental health disorders, leading to more effective therapeutic strategies.

Question 3

What is the primary goal of neuroscience?

Answer 3

The primary goal of neuroscience, is to understand the mind. That is, how we perceive, move,
think, remember. Or, more generally, what are the biological basis of our behaviour and our mental
wellbeing.

Question 4

What is neurocentrism?

Answer 4

So far, studies in the neurobiology of psychiatric and neurodevelopmental disorders have
focused on the role of neurons, seen as the only determinent of behaviour, a concept that we called
neurocentricsm.

Question 5

What is a different approach to mental health - an alternative to neurocentrism?

Answer 5

Another subtopic is the introduction of a different perspective, which has been developing over the
last ten to fifteen years, which sees glial cells in particular astrocytes as fundamental players in
determining brain function, behaviour and, as a consequence, mental health.

Question 6

What are two aspects of astrocyte function, which are important for the role in influencing
behaviour?

Answer 6

Astrocytes morphology and function were introduced in the previous subtopic.

1. So initially, we are going to look at astrocyte networks, and then, we’re going to move to
2. their role in the modulation of synaptic function, the so-called ‘tripartite synapse’

In part two, we are going to look at astrocytes in the pathology of the central nervous system,

Question 7

Do astrocytes modulate behavior?

Answer 7

Studies carried out in the past few years have led to the discovery that astrocytes can modulate
behaviour, and this has led to an increase in the interest in the potential causative on contributing roll
in psychiatric disorder.

We shall discuss this putative role of astrocytes in psychiatric disorder and
the difficulties that have been encountered in demonstrating this role and the cellular and molecular
mechanism involved, using studies on depression as an example.

Question 8

What is the tripartite synapse?

Answer 8

Tripartite synapse refers to the functional integration and physical proximity of the presynaptic membrane, postsynaptic membrane, and their intimate association with surrounding glia as well as the combined contributions of these three synaptic components to the production of activity at the chemical synapse.

Question 9

What does putative mean?

Answer 9

generally considered or reputed to be

Question 10

Who is Camillo Golgi? And when did he do his work with silver?

Answer 10

So in this first part, we shall look at how astrocytes may contribute to determine behaviour. This is
an image of astrocytes visualised originally in the 19th century with techniques, such as the silver
impregnation method, which was invented by Camillo Golgi in 1873.

Question 11

Do we actually have any evidence that astrocytes may indeed affect behaviour? Which study studied this and when?

Answer 11

This image is a micrograph of human astrocytes from a study that Han and colleagues performed
2013.

In an intriguing set of experiments, they transplanted human glial progenitor cells in
immunosuppressed mice.

These progenitors survived, they migrated long distances and that gave
rise to astrocytes with typical features of human ones, which are the ones that we see in red in this
picture.

Surprisingly, when Han and his colleagues examined the behaviour of these mice, they found mice
with human astrocytes perform better in learning tasks and displayed and improved long-term
potentiation, which is a strengthening of synaptic connection, which is thought to be the mechanism
underlying learning and memory.

Question 12

What four (glial cells) may be responsible for human cognitive abilities?

Answer 12

Ependymal, oligodendrocytes, astrocytes, microglia

Question 13

But how can astrocytes affect behaviour?

Answer 13

1. Well, astrocytes can obviously affect it indirectly, being, for example, involved in neuron and development and maintenance of a stable environment, which is
   their homeostatic role, which has been addressed in the previous subtopic. (Homeostatic role - A property of cells, tissues, and organisms that allows the maintenance and regulation of the stability and constancy needed to function properly.)
2. However, two function of astrocytes may potentially make them more directly responsible for behaviour in health and disease,
   and these are

a. the ability to release neurotransmitter, the so-called glial transmission and

b. their ability
to form astrocytic networks.

Question 14

What are the three elements that compose a synapse? (The “tri-partite”)

Answer 14

1. & 2. Two neuronal, the pre- and postsynaptic terminal belonging to two separate
   neurons and
2. an astrocytic process.

Question 15

What are the two features of astrocytes briefly that indicate a direct influence on behavior?

Answer 15

a. the ability to release neurotransmitter, the so-called glial transmission and
b. their ability to form astrocytic networks.

Question 16

What happens in synaptic activity?

Answer 16

During synaptic activity,

1. neurons release neurotransmitters.
2. An astrocyte responds to these
   neurotransmitters, which are represented in blue here.
3. We have elevations of calcium, and in turn,
4. they control neuronal excitability in synoptic transmission through calcium dependent release of glial
   transmitters, which are represented here in red.
5. The glial transmitter that astrocytes can release
   are glutamate, GABA, ATP, adenosine, D-serine, et cetera.
6. Probably every single transmitters
   that neurons can also release, and probably also express receptor transporter for all the major
   neurotransmitters.

Question 17

What does Homeostatic role mean?

Answer 17

A property of cells, tissues, and organisms that allows the maintenance and regulation of the stability and constancy needed to function properly.

Question 18

What did Han and colleagues, publish in 2012?

Answer 18

This is a diagram from an article by Han and colleagues, published in 2012. They perform some
experiments in mice. They were exploring the mechanisms underlying working memory impairment,
which is observed after cannabinoid exposure. In humans, one of the most significant consequence
of marijuana intoxication is in fact, an impairment in working memory. But the mechanism was so far
unknown.

In this study, they examined conditional mutant mice - that is mice that lack type 1 cannabinoid
receptor, CB1R in this slide, selectively either in brain astroglial cells or lacking CB1R in either
glutamatergic or GABAergic neuron.

Glutamatergic neuron release the excitatory neurotransmitter
glutamate, and GABAergic neuron release the inhibitory neurotransmitter GABA.

They exposed these different types of mice lacking the cannabinoid receptor either on astrocytes
or the two different type of excitator and inhibitor neurons acutely to cannabinoids.

They found an
impairment of spatial working memory in mice that lacked the CB1 receptor on glutamatergic or
GABAergic neuron.

But they found that preservation of this spatial working memory in mice that lack
the astrocytic CB1 receptor.

They also found that LTD - long term depression of synaptic strength - at
these hippocampal synapses was preserved in mice lacking the astrocytic CB1 receptor, and was
impaired in mice lacking the CB1 receptor in neurons.

Han and his colleagues explained their results assuming that cannabinoid exposure in vivo
sequentially activates astroglial cannabinoid 1 receptor.

This leads to a glutamate release from
astrocytes, which then will lead to activation of post-synaptic NR2B an MDA receptor, here in three,
which then elicits AMPA receptor, endocytosis.

This would result in working memory impairment.

It
has to be noticed that other authors has shown that CB1 receptor exists in astrocytes at this location
in the hippocampus, and they also show that they exist in the neurons in the presynaptic membranes
of glutamatergic and GABAergic neuron.

They also observed that the density of this receptor is 10 to 20-fold higher in GABAergic neurons
versus glutamatergic neurons.

They found that GABAergic and glutamatergic terminals containing
these cannabinoid receptors do synapse with dendrites and spines of pyramidal cells as indicated in
this diagram.

Activation of this presynaptic cannabinoid receptor reduces the release of glutamate
and GABA from glutamatergic and GABAergic neurons respectively.

However, since in the study
presented here by Hahn et al., they showed that cannabinoid could exert their actual working
memory also in the absence of the receptors neuron, they could conclude that astrocytes were
mediating their action.

Thee finding, presented here, supports the idea that astrocytes can play an
active role in cognition, and a role in its impairment in pathological state.

Question 19

How do astrocytes play a role in sleep?

Answer 19

Another behaviour that astrocytes might have a role in controlling is sleep.

For some time it has
been known that adenosine plays a role in controlling sleep homeostasis.

In particular, accumulation
of adenosine during wakefulness promotes sleep.

On the contrary, adenosine antagonist, such as
caffeine, notoriously promote wakefulness.

A number of studies have shown that the source of adenosine is astrocytes, and these regulate sleep
homeostasis.

Actually, as shown in this slide, astrocytes actually release ATP, and this is converted to
adenosine extracellular.

As we’ve seen, astrocytes can release gliotransmitter by different pathways,
including exocytosis.

The exocytotic release of chemical transmitter depends on the formation of a complex, which is
dependent on a protein called SNARE between vesicle and the target membrane.

It is possible to
genetically modify mice so that this SNARE dependent release of gliotransmitter is abolished.

These
are mice with the conditional astrocyte selective expression of the SNARE domain of the protein
synaptobrevin-2, the so-called dominant negative SNARE.

This genetic modification prevents both
tonic and activity dependent extracellular accumulation of adenosine, which acts on A1 receptor, as
shown here.

Studies in these transgenic mice incapable of exocytosis in astrocytes have demonstrated the role
for gliotransmission in the control of sleep.

Question 20

What does exocytosis mean?

Answer 20

a process by which the contents of a cell vacuole are released to the exterior through fusion of the vacuole membrane with the cell membrane

Question 21

What did Halassa and his co-workers discover?

Answer 21

In the words of Halassa and his coworkers, which made
this discovery, taken together these studies provide the first demonstration that the non-neuronal cell
type of the brain, the astrocyte, modulate behaviour and provide strong evidence of the important
role of A1 receptor in the regulation of sleep homeostasis and the cognitive decline associated with
sleep loss.

Question 22

How does the astrocytes’ organisation in astrocytic networks determine their influence on behaviour?

Gap Junctions.

connexin-30 and connexin-43.

biocytin

Answer 22

Astrocytes can be directly coupled with neighbouring astrocytes via gap junction, which form
aqueous channels between cells.

These gap junctions allow the passage of ions and small molecules
therefore they allow direct intercellular communication.

In astrocytes, gap junction are formed by
two proteins: connexin-30 and connexin-43.

It is possible to study the coupling of astrocytes into
networks injecting a soluable fluorescent dye in astrocytes, such as biocytin.

In this micrograph, this dye was injected in the astrocyte labelled with a white star, and diffused to all
astrocytes labelled in red in this figure, which the four are connected to the injected one in a network.

In green are astrocytes outside the network.

Gap junctions display selective permeability, and this
permeability can be regulated.

Furthermore, permeability is age-specific and region-specific.

Question 23

The previous slide we’ve seen how astrocytes may affect memory and controlled sleep. Does this
indicate that they may contribute to mental health pathology, considering that many mental health
disorders affect cognition and exhibit sleep co-morbidities?

Answer 23

perchance…

Question 24

What is the difference between AMPA and NMDA receptors?

Answer 24

The main difference between AMPA and NMDA receptors is that sodium and potassium increases in AMPA receptors where calcium increases along with sodium and potassium influx in NMDA receptors. Moreover, AMPA receptors do not have a magnesium ion block while NMDA receptors do have a calcium ion block.

Question 25

What happens when both connexin-30 and connexin-43 have been knocked out?

Answer 25

Gap junction can also be studied in
transgenic animals in which both connexin-30 and connexin-43 have been knocked out.

These animals
are unable to form functional gap junction.

Question 26

What do astrocytic networks do? How do they work?

Answer 26

Well, one important consequence of the coupling of astrocytes in
networks is the way in which this type of organisation allows for calcium rises, which are induced
by a neurotransmitter acting on to metabotropic receptor on the membrane of an astrocyte to
spread to connect the astrocyte, generating the so-called calcium waves, as shown here in the left
panel.

Combining these with the concept of gliotransmission, spreading calcium waves may cause
gliotransmitter release at remote synapses from the astrocytes which was originally activated.

In
the panel on the right, the role that astrocytic processes in blue could have at nearby glutamatergic
synapses in red is shown.

In A, only neuroglial interaction occurring at the three part of synapses are taken into consideration
- so just an astrocyte and a synapse.

The different steps involved in these dynamic interaction
are initially the first step, the release of neurotransmitter by the presynaptic neuron.

This
neurotransmitter will act on receptor and transporters in the astrocyte.

These will lead to the
release of gliotransmitter, which in turn can influence neuronal activity.

In addition to these three steps, glutamate that has been taken up by a neighbouring astrocyte, and
also the glutamate derivative glutamine, can diffuse and permeate through gap junction channels of
astrocytic networks, represented here by yellow stars.

This trafficking may result in the subsequent
release of gliotransmitter at the remote synapse, or even at the extrasynaptic sites, and hence,
affect the activity on the underlying neuronal network.

Question 27

What are gap junctions?

Answer 27

Gap junctions connect neighboring cells via intercellular channels that allow direct electrical communication as well as sharing of ions and small molecules (Figure 1) [1]. The channels are made of two hemichannels (one in each membrane) each consisting of six subunits known as connexins.

Question 28

What can astrocytic networks do?

Answer 28

Evidence is available that they can regulate the generation of a
rhythmic firing pattern in neuron.

This is necessary for several vital functions, such as respiration and
mastication.

Question 29

Rhett Syndrome and astrocytes

Answer 29

It is intriguing to note that respiratory rhythm is disrupted in Rhett Syndrome, which is an autism
spectrum disorder that is a neurodevelopmental disorder.

Active astroglial networks have been
also proposed to function as a master hub, which integrates the result of distributed processing
from several brain areas and support conscious states.

Question 30

What did Pereira and Furlan in 2010,

propose?

Answer 30

Indeed, Pereira and Furlan in 2010,
proposed that astrocytic network are essential for voluntary behaviour.

Only automatic behaviour
could be executed purely by neuronal network.

Dysfunction of these astrocytic networks, therefore, could lead to cognitive impairment.

It is
however, not clear if changes to the astrocytic network are cause or consequence of neuronal
dysfunction.

In part two, the potential involvement of astrocytic network dysfunction and depression
will be illustrated.

Question 31

What is a mental disorder?

Answer 31

According to the current edition
of the Diagnostic and Statistical Manual of Mental Disorders, DSM-5, which is used by clinicians
and researchers to diagnose and classify mental disorders: ‘A mental disorder is a syndrome
characterised by clinically significant disturbance in an individual’s cognition, emotion, regulation, or
behaviour that reflects a dysfunction in the psychological, biological, or developmental processes
underlying mental functioning.

Mental disorders are usually associated with significant distress in social, occupational, or
other important activities.

Examples of mental disorders include depression, bipolar disorder,
schizophrenia, autism.

One significant common feature of these disorders is that, in general, their
aetiology and pathophysiology are not fully understood.

However, we do think that a variety of genetic
and environmental factors are responsible for the onset.’

Question 32

Why do we still know so little about the potential role of astrocytes in psychiatric disorder?

Answer 32

It is very
difficult to study these in humans. Since the alterations may be subtle, people do not usually die of
psychiatric disorders and at the time of death, past history of the disorder may be ignored, other
illnesses may mask changes caused by psychiatric problem, alteration caused by pharmacotherapy
for the disorder may be indistinguishable from changes caused by the disorder itself.

The main line of evidence that are available to formulate hypotheses on the environment of
astrocytes in mental disorder derives from a range of studies in different part of system.

Question 33

What are three ways to study astrocytes and their function in mental health?

Answer 33

1. First type of study are human study, which are mainly postmortem.
2. The second type of studies are
   animal studies, including use of genetically modified animals.
3. The third line of evidence is in vitro
   studies, including astrocyte cultures, brain slices. For example, pharmacological studies on the effect
   of currently used therapies for a variety of neuropsychiatric disorders on glial cells, in vitro.

Question 34

astrocytes in

mental illness, and we shall particularly focus on major depressive disorder

Answer 34

We shall now examine some of the evidence currently available for an involvement of astrocytes in
mental illness, and we shall particularly focus on major depressive disorder. However, mention will be
made elsewhere of evidence available for schizophrenia.
Slide 6
Before discussing the evidence for a role of astrocytes in depression.

Question 35

What is depression?

Answer 35

It is a
common mental disorder that causes people to experience depressed mood.

It’s characterised by
loss of interest or pleasure, called anhedonia, feelings of guilt or low self-worth, disturbed sleep,
which can present as insomnia or excessive sleep, low energy, poor concentration.

Question 36

What are four neuropsychological processes underlying a depressed state?

Answer 36

There are a number
of theories on the neurophysiological processes underlying a depressed state, obviously, focused
on neuronal dysfunction, according to the neurocentric view.

1. And currently the dominant theory is
   the so-called monoamine hypothesis, which states that depression is the result of under activity of
   monoamine neurotransmitters, especially serotonin.

Indeed, most antidepressants aim at increasing
the level of available serotonin or monoamine neurotransmitters in general.

2. It is now also thought that dysfunction of the hypothalamic-pituitary-adrenal, HPA, axis, a system
   which is involved in the response to stress, may be implicated in the pathophysiology of depression.
3. Circadian rhythm abnormalities leading to disruption of sleep patterns have long been thought to
   play a role in mood disorders, including depression.
4. And finally, neurodegenerative and inflammatory
   alteration may also be contributing factors, particularly in late onset depression.

Question 37

What is pharmacological efficacy in depression?

Answer 37

Current pharmacological treatment has variable efficacy, but usually between 30 per cent and 50 per
cent of sufferers will not respond to a specific antidepressant medication, and about 50 per cent of
sufferers are poor responders to pharmacological treatment in general.

Question 38

So what evidence do we have that astrocytes may play a role in depression?

Answer 38

There are actually
several lines of evidence that suggests that astrocytes may play a role in depression and they are
derived from studies in animal model, studies on postmortem human tissue, and finally studies
on astrocytes in culture. And we’re now going to examine some example of each of these type of
studies.

Question 39

What do animal studies suggest about astrocytes in depression?

Answer 39

Let’s start with some example of studies in animal models.

In animal models of depression, an
astrocyte pathology is present.

Treatments that revert the astrocyte pathology also revert the
behavioural symptoms of depression in these models.

Chronic unpredictable stress is used as an animal model of depression. In this model, animals are
subjected for a number of days– 35 days in this case –to exactly the same sequence of 12 stressors,
two per day.

Example of stressors include cage rotation, light on, light off, cold stress, isolation,
crowding, cold swim stress, et cetera.

In order to assess the impact of chronic stress on astrocytes, the authors of this study measure the
level of messenger RNA for a specific marker of astrocytes, which is called glial fibrillary associated
protein, or GFAP, by a technique called in situ hybridisation.

The histogram in A gives the percentage of messenger RNA for GFAP - it’s percentage of control -
in animals kept in home cages, so not exposed to any stress, that are indicated here as CTR, and
animals exposed to chronic unpredictable stress.

The open bars indicate animals treated with saline, and we can see that there is a significant
decrease in the level of GFAP messenger RNA in animals exposed to chronic unpredictable stress.

This effect of stress can be reversed by injecting the animal with a glutamate modulating drug,
Riluzole.

At the bottom are representative autograph of the effect of chronic unpredictable stress on GFAP
messenger RNA expression compared to controls.

The authors of this study then employed a test, which is supposed to measure anhedonia in mice.

As
mentioned before, anhedonia’s considered a symptom of depression.

Rodents, some of mice, and
rats are born with an interest in sweet foods or solution.

Therefore, if given a choice between a water
bottle and a bottle containing a sucrose solution, mice should preferentially drink from the sucrose
solution containing bottle.

Reduced preference for sweet solution, in the sucrose preference test,
therefore, represents anhedonia, and chronic antidepressant can revert these reduced preference.

Day 15 of exposure to chronic unpredictable stress, stressed animals showed a significant decrease
in sucrose preference when compared to home cage control, CTR.

Disease presented in the panel
in A, where controlled animals are approximately three times more likely to drink from the sucrose containing
bottle than from the water bottle, and this preference is reduced in stressed animal.

The decreasing sucrose preference was even more significantly decreased in animal exposed for 35
days to chronic stress, and this is presented in the two open columns.

This decrease was reversed
by chronic Riluzole treatment in parallel with the decreasing glial pathology, which was observed
in the previous slides.

And these are the two columns in black, where both the control animals and
distressed animal presented a preference to drink the sucrose-containing water.

Question 40

Why does Riluzole reverse glial pathology and depressive behaviour?

Answer 40

It is proposed that Riluzole,
boosting glutamate uptake by astrocyte, would also boost glutamine production by astrocyte, and
this can be therapeutic in the context of depression.

Support from these hypotheses comes from studies which show that patients with major depressive
disorder exhibit reduced cortical levels of the neurotransmitter GABA.

And this is similar to rats,
which undergo chronic unpredictable stress.

Normalising GABA levels in these individuals correlates
with that clinical improvement.

GABA synthesis in neurons requires glutamine, which is produced
by the astrocytes.

Thus, it appears that the dysregulation of astrocytic support of the GABAergic
transmission contributes to the pathophysiology of major depressive disorder, and this process may
provide normal therapeutic targets for the treatment of this debilitating state.

Question 41

How do postmortem studies reveal astrocytes function in depression?

Torres-Platas et al.

measured levels of rna and protein for astrocyte specific marker GFAP in depressed suicide cases

Answer 41

We shall now move to some example of studies on human postmortem material.

In these studies by Torres-Platas et al., they measured the level of messenger RNA and protein for
the astrocytic specific marker GFAP in various brain areas of postmortem material obtained from
depressed suicides.

The authors looked at areas not involved in mood control, such as the primary visual cortex, on the
left, and the cerebellum, and areas known to be affected in mood disorders, such as the mediodorsal
thalamus and the coded nucleus, on the right.

Both GFAP messenger RNA and protein levels were found to be similar between control and suicide in
the visual cortex and the cerebellum.

However, both the messenger RNA and protein levels for GFAP
were significantly down-regulated in suicide in the mediodorsal thalamus and the coded nucleus
samples, indicating the presence of an astrocytic pathology, specifically in areas known to be involved
in mood regulation.

Question 42

What is GFAP?

Answer 42

Glial fibrillary acidic protein (GFAP) is the hallmark intermediate filament (IF; also known as nanofilament) protein in astrocytes, a main type of glial cells in the central nervous system (CNS). Astrocytes have a range of control and homeostatic functions in health and disease.

Question 43

How do studies conducted on cultured astrocytes relate astrocytes to depression?

Answer 43

We are now going to look at some examples of studies conducted on cultured astrocytes.

In these
studies, it was shown that various type of pharmacological and no pharmacological treatment for
depression can act directly on astrocytes.

In this first study, the authors looked at the effect of a commonly used antidepressant, Fluoxetine,
better known as Prozac, on cultured astrocyte.

Prozac is one of the most famous and used antidepressant medication.

Its mechanics of action is
supposed to be the inhibition of serotonin reuptake in the brain.

This would increase the amount of
serotonin present and, consequently, serotonergic neurotransmission.

However, in the experiment
presented here, the authors showed that Prozac can also act directly on astrocytes, and this is
shown in the panel at the top.

In black are control astrocytes and in light grey, Fluoxetine-treated
astrocytes.

As it can be seen, the production of a number of trophic factors is increased in astrocytes treated
with Fluoxetine.

The production of BDNF, VEGF, and VGF are all increased in Fluoxetine-treated astrocytes.

Interestingly, this effect is totally independent of serotonin, since treating the cultured astrocytes with
serotonin does not induce trophic factors synthesis, and this is presented in the panel at bottom.

In the panel on the right, we can see that serotonin treatment of the same cultured astrocyte had no
effect on the synthesis of neurotrophic grow factors.

It is interesting to note here that the full therapeutic effect of Prozac may be delayed until four to six
week of treatment.

This has proved difficult to explain if Prozac acts via increasing serotonin level at
the synapse, because the effect should be almost immediate.

However, if the main effect of Prozac is
on astrocytes via induction of the production of trophic factors, the therapeutic delay may be easier
to explain, since it would take some time for the increasing trophic factor to lead to increasing uptake
plasticity, neurogenesis, and restoration of damaged neuronal network.

Question 44

What about a causal link between astrocyte pathology and depression, or clearly
suggest what the underlying mechanism might be?

Answer 44

The evidence presented, so far, suggests the presence of a glial pathology, probably astrocytic
atrophy in depression, which correlates with the presence of depressive symptoms in humans and
animal models.

The studies that I’ve shown on astrocytes in culture, treated with antidepressant, also suggest that
potentially the therapeutic effect may be due to action on glial cell.

However, none of the studies
discussed so far, can confirm a causal link between astrocyte pathology and depression, or clearly
suggest what the underlying mechanism might be.

This particular issue was addressed in further studies, utilising again postmortem material and
animal models, and we are going to examine some of those.

We saw earlier how astrocyte can be connected in networks, which are not fixed, but can be
modulated.

We also mentioned how these networks can play a role in integrating neuronal activity.

Question 45

Is
it possible that disruption of astrocytic networks may play a role in depression?

Ernst and co-workers

Answer 45

Let’s look at some evidence from postmortem human material and animal models of depression.

Some evidence for postmortem material suggest that disruption of astrocytic networks may indeed
play a role in depression.

As we mentioned in part 1, astrocytes are connected in networks via gap junction, which are
composed by the proteins connexin 30 and connexin 43.

Ernst and co-workers measured the levels
of connexin 30 and connexin 43 in the prefrontal cortex of suicides, suffering from a range of
psychiatric disorders.

The graph of the top refer to the results for the messenger RNA of connexin
30 in A and connexin 43 in B.

And the graphs at the bottom refer to the protein levels, again of
connexin 30 the left and 43 to the right.

And the images in the upper right corners of the top graph
illustrate semi- quantitative PCR data from control and suicide case.

What is apparent from these graphs is that both, the level of messenger RNA of connexin 30 and
connexin 43, and the level of connexin 30 and connexin 33 proteins are reduced in the suicide
completers versus control subjects, and this suggests a dysfunction of astrocytic networks in
depressed individuals.

Based on the findings of the pilot study on suicide completer shown in the previous slide, the level of
connexin messenger RNA and protein were then examined in rats that had been exposed to chronic
unpredictable stress, which as we have seen is a model of depression.

As in humans, the levels of
messenger RNA and protein of both connexins were found to be decreased in the prefrontal cortex
of stressed rats.

In the panelling A, we have the results for protein levels. In the panel on the right, we have the result
for messenger RNA levels.

The protein levels were determined by western blot, and in A, we have
representative western blot images for connexin 43 and beta-actin, used as a loading control.

The first three column in A, presents results from controlled rats, either untreated or treated, with
two different antidepressants, and columns four to six present results from rats exposed to chronic
unpredictable stress and treated in column four or treated with two different antidepressants in
columns five and six.

Results are presented in the same order for messenger RNA level in C.

What is apparent, is that, in stressed rats, both protein and messenger RNA level for connexin
43 were significantly decreased.

The levels were restored by treating with either of the two
antidepressants, either Fluoxetine or Duloxetine. It is apparent that both the level of connexin
43 protein and the level of connexin 43 messenger RNA were significantly reduced in chronic
unpredictably stressed rats.

At the same time, both the level of connexin 43 protein and the level of
connexin 43 messenger RNA were restored to normal by antidepressant treatment.

Question 46

What are the consequences of the decreased level of connexin protein?

Answer 46

Lucifer yellow, which is dye, was injected into the rat brains, as illustrated here.

As you can see
from the graphs at the top, which are quantifications of the image at the bottom, both the diffusion
distance and the number of coupled cells were reduced by chronic unpredictable stress, and this
effect was reversed by antidepressant treatment.

In the micrograph at the bottom, we see in CTR, CF, and CD are three control groups: untreated
on the left or treated with the two antidepressant on the right.

And we can see that the distance of
diffusion of the dye, in green, is similar in all three panels.

The bottom left panel, CUS, shows a much reduced, both distance of diffusion and number of couple
cells, which is restored to normal in SF and SD, which are the two chronic unpredictable stress group
with antidepressant treatment.

Could the decreased number of gap junctions and a reduced coupling of astrocytes have a causative
effect in depression?

The authors infuse the chemical carbenoloxone, which blocks gap junction in the
prefrontal cortex.

The asterisk in A indicates the point of the infusion.

Injection of carbenoloxone in the prefrontal cortex induced depressive-like behaviour in rats.

This
was measured by using the sucrose preference test, which we’ve already spoken earlier.

The first
column is animal treated with Vehicle, and shows the effect of carbenoloxone infusion on the sucrose
preference test.

Animals that had a control infusion of phosphate-buffered saline, displayed a preference for sucrose containing
water.

This was decreased, indicating depressive-like symptoms already at the lowest
dose of carbenoloxone.

But with increased doses, the depressive symptoms became more apparent.

Question 47

Summary of the studies:

Answer 47

To summarise the studies described here, we’ve seen how decreased level of connexin, both
connexin 30 and connexin 43, which are the components of gap junction in astrocytes, were identified
in both major depressive disorder and in experimental stress.

Furthermore, both Fluoxetine and other antidepressant can reverse these changes in animal models.

We’ve also seen how blocking gap junction in the prefrontal cortex is sufficient to induce depressive
behaviour in animals, suggesting that astrocytic dysfunction may be sufficient to induce the onset of
depression.

However, the mechanism whereby dysfunctional astrocytic networks may affect mood
have not been established yet.

We then hypothesised that decreased expression of connexin 30 and connexin 43 may alter calcium
wave propagation and communication between astrocytes, possibly leading to a decrease in the
simultaneous release of gliotransmitter and/or affecting the metabolic roles of astrocytic networks,
such as potassium buffering, supply of energy substrate, et cetera.

Therefore, astrocytic network
dysfunction may play a role in depression.

Question 48

But what about gliotransmission?

Answer 48

Whilst the role for gliotransmission in depression can be hypothesised, since disruption of astrocytic
networks could affect neurotransmission, currently we have no direct evidence to support this
assumption.

On the other hand, some recent study on sleep deprivation and depression seem to
support the idea, overall, for gliotransmission in depression.

As you may remember, from part one, astrocytes have been shown to play a role in the regulation
of sleep via SNARE-dependent signalling, which is mediated through the adenosine A1 receptor on
neurons.

Sleep deprivation is a potent short term antidepressant, which is effective in approximately 60 per
cent to 70 per cent of the patient.

Sleep deprivation, in the form of one or more nights of total sleep
deprivation, can rapidly alleviate symptoms of depression.

Interestingly, in learning what happens in
human depressed patient, sleep deprivation can reduce depressive-like symptoms in mouse models
of depression.

But this reduction in depressive-like symptoms is not observed in mice in which the
vesicular release of gliotransmitter is prevented by genetic manipulation, or if adenosine signalling is
prevented using an adenosine receptor antagonist, or knocking out the A1 Receptor gene.

This suggest that anti-depressive effect of sleep deprivation require gliotransmitter release from
astrocytes.

This suggests an involvement of gliotransmission in depression.

Question 49

What is SNARE-dependent signalling?

Answer 49

The primary role of SNARE proteins is to mediate vesicle fusion – the fusion of vesicles with the target membrane; this notably mediates exocytosis, but can also mediate the fusion of vesicles with membrane-bound compartments

Question 50

So astrocyte pathology may play a role in depression, but what causes astrocyte pathology in
depression?

Answer 50

Stress, acting on the HPA axis, may be a causative factor in depression, and acute
and chronic stress may alter astrocyte morphology and physiology.

Most of this alteration can be
prevented by antidepressant and other protective treatment.

Further, correlative evidence supports
a role for astrocytes in most, if not all, psychiatric disorder, and you can review some of the evidence
in the key readings.

Question 51

Conclusion of Part 1 Week 2

Answer 51

In conclusion, much circumstantial evidence supports the hypothesis, overall, for astrocytic
dysfunction in psychiatric disorder.

And this evidence comes from a range of studies, as we have
seen, from studies in humans to studies in cell culture.

Often, the most obvious astrocytes pathology
appears to be astrocytic atrophy.

It is currently impossible to determine whether astrocytic pathology is the primary cause of
psychiatric disorders, such as depression or schizophrenia, or whether such pathology always
emerges as a consequence of neuronal dysfunction - that is, it is secondary to neuronal pathology.

Even if genetic studies would suggest that, at least in some cases, astrocytic pathology may be the
primary cause, since in some animal models genetic alterations, which are limited to the astrocytes,
are sufficient to generate mood disturbances, and you can find evidence of these in the section on
schizophrenia.

Targeting astrocytic pathology appears, in some cases, to be sufficient to ameliorate behavioural
disturbances, regardless of whether astrocytic pathology is the primary cause of the disorder or
secondary to neuronal dysfunction.

Since a considerable body of evidence now suggests that neurons are not the sole determinants of
behaviour, it seems fundamental that future research into the neurobiology of psychiatric disorder
shifts this focus from a neurocentric perspective to, at least, a neuroglial one, if not affirming a
gliocentric one, as suggested by some researchers.