Glu, Stroke & SZ Flashcards
(194 cards)
1
Q
What percentage of Marmite and Parmesan are made up of glutamate?
A
25%
2
Q
What brings Umami flavour about?
A
Glutamate activated receptors on the tongue
3
Q
What percentage of all CNS neurons are glutamate?
A
60-70%
4
Q
What percentage of all CNS neurons are GABA?
A
30%
5
Q
What percentage of all CNS neurons are ‘Neuromodulators’?
A
<0.1%
6
Q
What does neurome mean?
A
Neurons in terms of their numbers
7
Q
How many dopamine neurons are there in the VTA and SNpc?
A
400-600 x 10^3
8
Q
How many 5-HT neurons are there in the raphe?
A
300 x 10^3
9
Q
How many ACh neurons are there in the nucleus basalis Meynert?
A
200 x 10^3
10
Q
How many noradrenaline neurons are there in the locus coeruleus?
A
20-50 x 10^3
11
Q
What is the precursor to GABA?
A
Glutamate
12
Q
What converts glutamate to GABA?
A
GAD
13
Q
What is the precursor to glutamate?
A
alpha-ketoglutarate
14
Q
What converts alpha-ketoglutarate into glutamate?
A
Aminotransferase (is the result of metabolism, generating high levels of glutamate)
15
Q
Besides GABA, what are three other metabolic results of glutamate?
A
- Glutamine
- Proline
- Arginine
16
Q
What enzyme causes glutamine to recycle as glutamate?
A
Glutaminase
17
Q
What pathway of the TCA cycle causes synaptic glutamate?
A
Glutamine to glutamate via glutaminase
18
Q
What formation of receptors does glutamate have?
A
Tripartite synapses- three membered arrangement
- Presynaptic
- Postsynaptic
- Astrocytic processes
19
Q
What is the major purpose of astrocytic processes in the glutamate uptake and transport?
A
They regulate the extracellular space and maintain a normal concentration here
However, they are not overly efficient, so the extracellular fluid can activate as its concentration level
20
Q
What is the extracelular concentration level of L-glutamine?
A
1 microM
21
Q
What is the intracellular concentration level of L-Glu?
A
10 milliM
22
Q
What is the concentration of vesicular L-Glu?
A
100 milliM
23
Q
How is the action of glutamate in the synapse terminated?
A
- Uptake on the presynaptic terminals
- Uptake using astrocytic processes
24
Q
What channels are used to drive the uptake of glutamate?
A
- K, Na channels
- Sometomes is against the concentration gradient of glutamate
25
What occurs when glutamate is taken into the astrocyte?
- Convert glutamate to glutamine
- Glutamine is safely free to leave
- It is then taken up by presynaptic terminals
- Glutamine-glutamate shuttle
26
What is the most major source of glutamine as a neurotransmitter?
Glutamine-glutamate shuttle
27
What happens if the uptake/transport of L-Glu becomes perturbed?
Causes malfunction and excessive L-Glu release
28
What are some important transporter for extracellular glutamate?
EAAT1-5 (Excitatory Amino Acid Transporter family)...
- GLAST- Glutamate ASpartate transporter (EAAT1)
- GLT-1- Glutamate transporter (EAAT2)
- EAAC1- Excitatory Amino Acid Carrier (EAAT3)
29
What are two transporters important in shuttling glutamate out of the astrocyte?
- SN1- System N transporter
- SA1- System A transporter
30
What is an important intracellular L-glutamate transporter?
VGLUT1-3- Vesicular glutamate transporters
31
What do ionotropic Glu-R channels form?
- Receptor-ionophore complex
- Ligand-gated non-selective cation channel
32
When glutamate is bound to an ionotropic receptor, how many cations can pass?
Up to 3 depending on certain factors
Erev = 0mV
33
Where do post-synaptic ionotropic glutamate receptors reside and what do they do?
- Reside as a post-synaptic density opposite active sites
- They bind to glutamate causing ion flow of an inward current and therefore depolarisation
34
Outline NMDA-R pharmacology
- Specific agonist (for NMDA)
- Specific antagonist (D-AP5), If this has an effect, an NMDA receptor is involved
35
Outline AMPA-R pharmacology
- Non-NMDA-R
- Non specific for AMPA and Kainate but prefers AMPA
- Specific antagonist for non-NMDA-Rs (NBQX)- blocka both AMPA-Rs and Kainate-Rs
36
Outline Kainate pharmacology
- Non-NMDA-R
- Non specific for AMPA and Kainate but prefers kainate
- Specific antagonist for non-NMDA-Rs (NBQX)- blocka both AMPA-Rs and Kainate-Rs
37
What are AP5 and NBQX?
Competative antagonists (bind to the same site as the agonist (glutamate site))
38
How does NMDA-R react to calcium?
- Highly permeable to Ca
- Significant contribution to Erev at physiological concentrations (equilibrium reversal potential)
- Ca permeability 13 x Na
- Jahr & Stevens 1993
39
What is the principle action of NMDA channels?
To generate calcium levels intracellulary
40
How do NMDA-Rs contribute to fast excitatory neurotransmission?
- Under low levels of activation, the primary release is for the AMPA receptor
- When significant depolarisation occurs and sustained action- NMDA receptor is also activated
- More calcium enters and therefore more depolarisation causing plastic changes (postsynaptic)
41
Outline NMDA-Rs functional properties
- Postsynaptic- depolarisation (Erev=0mV, Na, K)
- Voltage dependent Mg block, so hyperpolarised membrane potential but when there is depolarisation is removed and can flow
- On autonomic presynaptic receptors it augments neurotransmitter release
- Slow kintetics
- Highest Ca permeability
- Glycine co-agonist
42
Outline AMPA-Rs functional properties
- Postsynaptic- depolarisation (Erev=0mV, Na, K)
- Not presynaptic (represent a good indicator of postsynaptic effect)
- Fastest kinetics
- Lowest permeability for Ca
43
Outline Kainate-Rs functional properties
- Postsynaptic- depolarisation (Erev=0mV, Na, K)
- Presynaptic action modifying release through augmentation and suppression
- Intermediate kinetics
- Intermediate Ca permeability
44
What are the three types of metabotropic receptor?
- Group 1
- Group 2
- Group 3
45
What G-proteins are group 1-Rs linked to?
Gq
46
What G-proteins are group 2-Rs linked to?
Gi/0
47
What G-proteins are Group 3-Rs linked to?
Gi/0
48
Outline Group 1-Rs functional properties
- Mainly postsynaptic
- Depolarisation/increase excitability
- Close K channels
Enhance NMDA-R mediated currents
49
Outline group 2-Rs functional properties
- Mainly presynaptic
- Decrease release
- Downregulate Ca channels
50
Outline group 3-Rs functional properties
- Mainly presynaptic
- Decrease release
- Downregulate Ca channels
51
What happens if you add group 1 mGluR to NMDA?
- If add DHPG it augments the size of NMDA depolarisation
- So is more likely to contribute during sustained synaptic activity
52
What occurs if you add AP4 to a neuron?
Is selective for Group 3 mGluR causing regulation of synaptic function and depression of EPSP
53
What occurs if you add DCG-IV to a neuron?
Is selective for Group 2 mGluR causing regulation of synaptic function and depression of EPSP
54
What do autoreceptors do for glutamate release?
Control L-Glu release by L-Glu
55
What do heteroreceptors do?
Regulate release of other neurotransmitters (e.g. GABA)
L-Glu spill-over to adjacent L-glu or GABA synapses
56
Where are ionotropic channels located on the postsynaptic membrane?
Either within the postsynaptic density or outside of it
57
Where are metabotropic channels located on the postsynptic membrane?
Outside of the postsynaptic density
58
What is the gene family for AMPA and what is its percentage?
GluA1-4 (56-73%)
59
What is the gene family for Kainate and what are their percentages?
- GluK1-3 (75-80%)
- GluK4-5 (68%)
Overall 45%
60
What is the gene family for NMDA and what are their percentages?
GluN1-3:
- GluN1
- GluN2A-2D (38-53%)
- GluN3A-3B (50%)
Multiple spliced variants
61
What is the homology between iGlu-R gene families?
18-40%
62
What is the gene family for Group 1 (ACPD) and what is its percentage?
mGlu1 and 5 (62%)
63
What is the gene family for Group 2 (ACPD) and what is its percentage?
mGlu2 and 3 (68%)
64
What is the gene family for Group 3 (L-AP4) and what is its percentage?
mGlu4, 6, 7, 8 (69-74%)
mGlu6 only in the retina
65
What is the homology between the mGlu-R gene families?
42-45%
66
What shape are iGlu-R channels and how many binding sites doe they have?
Tetramers with 4 agonist binding sites- all 4 have to be full for activation
67
What is the formation of the NMDA receptor?
- Hetromeric only (dual agonism)
- Usually 2 GluN2 (glutamate binding)
- 2 GluN1 (glycine binding)
Laube 1998
68
What is the formation of AMPA and Kainate receptors?
- Homomeric or hetromeric
- However, GluK4/5 heteromeric inly, must be with GluK1-3
Rosemund 1998
69
What is the dual mode of action for Kainate-Rs?
- Hybrid contribution of ionotropic and metabotropic heteromers for functioning
- GluK2 and GluK5 (GluK5 interacts with a GPCR)
Melyan et al 2002
70
What is the structure of mGlu-R?
- Large extracellular amino acid terminal with 'venus fly trap' binding region
- Cys rich domain
- 7 transmembrane domains
- intracellular carboxy terminal
- G proteins binding to intracellular loops 2 and 3
71
When are mGlu-Rs functional?
Homo-dimers
Linked by disulphide bright (s-s) between VFT domains
72
When was the toxic effect for L-Glu first reported?
- Lucas and Newhouse 1957
- Systemically injected MSG into young mice P2-16 led to inner retinal degeneration and complete cell loss within 2 weeks
73
What did John Olney do following concerns about MSG?
- 1969
- Concerns about MSG as a flavour enhancer in baby food
- Brain damage demonstrated in new-born mice and primates following systemic (suncutaneous) administration of MSG
74
What are some amino acids that cause necrotic cell death?
- Olney 1971
- L-Glu
- L- Aspartate
- NMDA
75
When was the term neurotoxicity switched to excitotoxicity?
When out understanding of the role of L-Glu and iGluRs in excitatory neurotransmission increased in 1980s
76
What are 3 types of excitotoxic processes described in vitro?
- Acute - 1-3 hours
- Delayed - 2-12 hours
- Slow - 24-72 hours
77
What do the types of excitotoxic processes in vitro depend on?
- Receptor subtypes involved
- Level of activation
- Cell type as they express different iGlu-Rs and mGlu-Rs
78
What occurs during acute (swelling-induced) L-Glu excitotoxicity?
- S.Rothman 1985
- Leads to rapid cell death in 1-3 hours: necrosis
79
What is necrosis?
- Cell breaks up
- The membrane bursts
- The intracellular contents are lost
80
What is the pharmocology during acute (swelling-induced) L-Glu excitotoxicity?
- Induced by high levels of e/c L-Glu (0.5-1microM) applied up to 30 mins
- Reduced by NMDA rather than non-NMDA receptor antagonists (mainly NMDA-R mediated)
81
What is the mechanism during acute (swelling-induced) L-Glu excitotoxicity?
- Non-NMDA receptors are not involved as non-NMDA-R are desensitised due to duration
- Although NMDA receptors are Ca permeable, no a Ca dependent process (if remove extracellular Ca from the medium, it does not stop the effect from occuring)
- Depolarisation due to Na and Ca influx via receptor ligand gated and voltage gated ion channels and passive Cl influx via Cl channels (because the balance of Cl is dependent on the membrane concentration of the system)
- The cells load with anions and cations changing the osmotic concentration (increase, so H20 enters down osmotic graident via aquaporins) and causing oedema
- Mitochondrial collapse and the membrane can no longer contain the pressure of oedema so underfo cell lysis
82
What occurs during delayed (Ca dependent) L-Glu excitotoxicity?
- D.choi 1987
- Leads to delayed cell death after 6-12 hours: a hybrid of apoptosis and necrosis
83
What is the pharmacology during delayed (Ca dependent) L-Glu excitotoxicity?
- Induced by high levels of e/c L-Glu (0.5-1microM) applied <5min. Neurons recover from acute swelling
- Reduced by both NMDA and non-NMDA antagonists applied 2-8 hours after L-Glu application
84
What is the mechanism during delayed (Ca dependent) L-Glu excitotoxicity?
- All iGlu-Rs involved
- Ca dependent with a prolonged rise in intracellular Ca from the influx through NMDA-Rs (and probably some Ca permeable AMPA/Kainate-Rs) (If remove Ca in EF can block)
- Probably a self-propogating/sustaining process. First wave of cell death initiates further L-Glu relese, there is more excitotoxic damage and cell death and so on (dead, burst cells release their contents affecting their neighbour)
- 'Delayed' antagonist application effective- could be useful in clinical setting (similarities with progressive neuro-degeneration seen in disease states in vivo)
85
What occurs during slow excitotoxicity?
- J.A Larn et al 1997
- Cell death after 1 or more days in vitro- apoptosis
86
What is the pharmacology of slow excitotoxicity?
-Induced by prolonged exposure to AMPA or kainate receptor agonists for 24-72 hours
- Blocked by CNQX but not MK-801, so non NMDA not NMDA receptor mediated
87
What is the mechanism of slow excitotoxicity?
Probably Ca dependent, with a prolonged rise in intracellular Ca from the influx through Ca permeable AMPA/Kainate receptors or voltage gated Ca channels
88
What is the TUNEL staining method?
- Method for identifying where DNA has fragmented during apoptosis
- Labels neurons so is a signature stain for apoptosis
89
See slide 33 of lecture 4 to be confused
90
When Kainate is injected into a cerebral cortex neonate rat what morphologies do we see?
- Apoptotic cells
- Intermediate hybrid cells
- Necrotic cells
- Martin LJ et al 1998
91
Outline what is seen in an apoptotic cell undergoing excitotoxic damage in vivo
- Highly dense nucleus and **round clumps of chromatic**
- Intact nuclear and plasma membrane
- Condensed cytoplasm
92
Outline what is seen in an intermediate hybrid cell undergoing excitotoxic damage in vivo
- Nucleus is irregular with partially condensed clumps of chromatin
- Intact nuclear and plasma membrane
- Many vacuoles in the cytoplasm
93
Outline what is seen in a nectrotic cell undergoing excitotoxic damage in vivo
- Nucleus has loose irregular aggregates of chromatin (highly fragmented- does not condense)
- Dissolution of membranes
- Cytoplasm into many vacuoles
94
What is the definition of (neuro)excitoxicity?
Over-activation of iGLu-Rs triggering cell death via necrosis and/or apoptosis
Olney
High levels of L-Glu not toxic per se, could also stop the activation of receptors or not just due to excessive release of endogenous L-Glu
95
Besides from iGlu-Rs causing excitotoxicity what other receptor contributes?
- mGlu-R only Group 1 which acts facilitatory or permissive role
- Unlikely group 2 or 3 as they are inhibitory so would help slow the process down
96
What are 4 possibilities of increased endogenous L-Glu release?
- Ischaemia Stroke/Cardiac arrest- restricted blood flow to brain regions, O2 deprivation and run down of metabolic energy sources which is essential for ion gradients
- Hypoxia- O2 deprivation (asphyxiation, drowning)
- Epilepsy- also associated with excessive L-Glu release and so depending on the severity, can cause excitotoxic damage and so lead to progressive deterioration of condition
- Neurodegeneration- necrotic cell death and release of L-Glu leads to a vicious cycle of excitotoxic damage
97
How can the prescence of exogenous iGlu-Rs cause excitotoxicity in vivo?
- Glutamate is highly controlled crossing the BBB but some substances mimic endogenous L-Glu action
- Ingestion of environmental toxins present in food
98
What percentage of strokes are ischaemic?
85%
99
What percentage of stroke are haemorrhagic?
15%
100
By the age of 75, how many people will suffer from a stroke?
- 1 in 5 women
- 1 in 6 men
101
What is an ischaemic stroke?
- Blockage of blood vessels
- Thrombotoc or embolic small artery occlusion
- Blood flow is arrested
102
What is a haemorrhagic stroke?
- Rupture of blood vessels
- Intracerebral (hypertensive) haemorrhage, subarachnoid haemorrhage (ruptures aneurysms)
103
What does a CT scan of a Ischaemic stroke look like?
- Cerebral infarct- dead or necrotic tissue that results from the disrupted blood supply.
- On the scan the dark area shows a lack of blood flow and irrigation of the tissue
104
What does a haemorrhagic stroke look like on a CT scan?
- Clear white area where fluid is leaking into and displacing the brain tissue
- Darker rim around white area showing oedema
105
What is a Mini (minor) ischaemic stroke?
- Mild stroke
- Transient ischaemic attack of a brief episode (minutes to hours) of neurological dysfunction
- Resolves in 24 hours
- No evidence of infarction pathology
106
What is a major ischaemic stroke?
- Temporary loss of function >24 hours
- May resolve in up to 4 weeks- reversible ischaemic neurological defecit (RIND)- little evidence of pathology
- Permanent loss of function reflects the extent of the cerebral infarct
107
Explain how extracellular glutamate is involved in stroke
- Processes controlling EC glutamate use Na and K gradients to get the cellular elements to take up glutamate against a concentration gradient
- EC is normally 1 microM of Glu so if these uptake mechanisms fail, it will quickly rise
- Require high K inside and high Na outside
108
Explain the impact of ischaemia on ionic transport function
- Reduced blood supply means less ATP:ADP levels in neural cells
- Na/K/ATPase less effective
- Ion gradients and equilibrium potentials for Na and K reduced causing a feed forward effect
- Causes depolarisation of presynaptic terminals and therefore vesicular L-Glu release
- Reversal of L-Glu transporter mehanisms and L-Glu potential being the driving gradient, further exacerbating L-Glu release
109
Where in animal models was an ischaemia replicated?
- Inclusion of the middle cerebral artery (key brain vasculature supply)
- Is the first point where vessels are narrower (loss of motor function)
110
How were animal models of ischaemia created?
Insert a filament into the middle cerebral artery where it leaves the carotid supply
111
What was 'Merks new wonder drug'?
MK-801 (dizocilpine)
Very effective due to potency, could co-apply to prevent excitotoxic damage
112
What was the IC50 of MK-801?
140nM
113
What is the IC50 of D-AP5?
3-10microM
114
What are 4 issues with MK-801 and why was development stopped?
- Needed to be given immediately after artery occlusion (narrow therapeutic window)
- High doses required for protection (low therapeutic index- limits ability to avoid toxic effects)
- Side effects in humans could be significant and severe: memory impairment, potential induction of SZ-like psychosis
- MK-801 neurotoxic syndrome in rodents- vacuolization and mitochondrial breakdown causing death of CNS neurons
115
Why could NBQX not be used as an AMPA-R antagonist clinically?
Has poor water solubility so could not be administered systemically
116
What is a new AMPA-R antagonist?
YM872/Zonampanel
117
Why was zonampanel promising in treating ischaemic strokes?
- Reduces MCAO infarct volume in rats by 50-60%
- Effective when given up to 3 hours after stroke
118
Why did zonampanel fail clinical trials?
- At phase 3
- Lack of efficacy
119
What is an additional AMPA-R antagonist that failed for the same reasons as zonampanel?
Talampanel
120
What was a potential drug for paramedics to administer within the 'golden hour' of a stroke?
- Intravenous Mg within 2 hours of the onset of symptoms (neuroprotective treatment for epilepsy)
- But has been shown to be ineffective in reducing long-term disability in stroke (Saver et al 2015, Shirkova et al 2017)
121
What are problems if L-Glu-R mediated transmission is suppressed over a long time?
Problems with side-effects and cognitive deficits
122
What happened to an 8-day old rat when blocked NMDA-Rs?
- Used TUNEL staining method
- Saw cell death, apoptotic profiles
- Is toxic
123
What are the distinct roles for NMDA-Rs based on GluN2 (NR2) subunit composition in neonates?
- NR2B (GluN2B) synaptic receptors will be located within the post-synaptic density
- Is for singalling intracellular Ca
124
What are the distinct roles for NMDA-Rs based on GluN2 (NR2) subunit composition in adults?
- Overtime, NR2A displace NR2B to locations outside the synapse
- NR2A (GluN2A) in the synapse are important for synaptogenesis, AMPA-R insertion and spine proliferation also increasing IC Ca for CREB
- NR2B are then extrasynpatic causing LTD, AMPA-R endocytosis, detecting environmental glutamate and influencing IC Ca
125
Which NR2 subunit is more affected by NMDA induced excitotoxicity?
- NR2B (GluN2B)
- As is more in post-natal brain, more susceptable (Zhou and Baudry 2006)
126
What does Ca influenced CREB cause?
Increase of BDNF and Bcl-2 (anti-apoptotic factor that prevents mitochondrial permeability changes that trigger apoptosis)
Survival/neurogenesis
127
In addition to Ca CREB what other to Ca dependent factors does NR2B produce?
- Calpains- excessive activation and protein breakdown
- NO- Cell damage
128
Besides targetting GLu receptors what is another therapeutic strategy?
Augment the remaining L-Glu synaptic function and limiting long-term consequences of insults such as reperfusion injury and neuroinflammation
129
Why is there a mismatch between DA antagonist receptor binding and effects adding a flaw in the dopamine hypothesis for SZ?
- Locomotor effects (sedation and reduced motor activity) are very quick (mins)
- Antipsychotic effects take longer- weeks
130
What are 5 flaws in the dopamine hypothesis for SZ?
- Schizophrenics with negative symptoms respond poorly or not at all to treatment with 'typical' fist generation antipsychotics
- Schizophrenics develop hyperfrontality, associated with poor working memory (regional blood flow during WM task using 133Xe dynamic SPECT)
- Made worse with typical antidepressants
131
What is hyperfrontality?
Reduced function of the frontal lobe of the neocortex
132
What is proof of dopamine antagonism (antipsychotic) being bad for schizophrenics?
- Daniel et al 1989
- Apomorphine (DA agonist) reversed the reduction in cerebral blood flow associated with hyperfrontality in schizophrenics
- Due to a decrease in dopaminergic neurotransmission in prefrontal areas in SZ.
133
What does PCP stand for?
Phencyclidine
134
What was the original purpose for PCP?
- Hailed as a new wonder drug
- Produce dissciative anaesthetic state- loss of pain perception but subject remains conscious
135
What do acute doses of PCP produce?
- Auditory hallucinations
- Disorientation
- Paranoia
- Panic and intense aggression
- Potentiation of symptoms in schizophrenics
136
What is the effect of chronic PCP administration in primates?
- Vervet monkeys (0.3mg/kg) for 2 weeks
- Develop persistent cognitive deficits consistent with negative symptoms of SZ
- Hyperfrontality
- Lowered dopamine utilisation in dorsolateral prefrontal and prelimbic cortex but not the nucleus accumbens
- Ameliorated by atypical antipsychotic clozapine treatment
- Jentsch et al 1997
137
What recptors/channels is PCP active at?
- **NMDA-R antagonist**
- NE/DA/5HT
- Mu and delta opiate
- K/Na/nAChR/mAChR/AChE
- GABA
138
How does PCP create psychosis?
It blocks NMDA receptor channels at a dose that also produces psychosis in a non-competative fashion
139
What are three classes of NMDA-R antagonists?
- L-Glu site (competative)- D-AP5
- Glycine site (competative)- 5,7-DCKA
- Open channel blockers (non-competative)- Ketamine, PCP, MK801
140
When are open channel blockers of NMDA-Rs active?
- These blockers are only effective if the channel is open such as undergoing activation by an agonist
- As a result, the block is use-dependent- the more the channel is activated, the greater the block
- This is because they bind within the pore region (deep in the channel)
141
What forms the glutamate/NMDA receptor hypofunction hypothesis (3)?
- Acute NMDA receptor ion channels blockade (MK-801, PCP, Ketamine) causes a psychotic state in humans
- Long term administration of these leads to pathology in the PFC similar to SZ- also areas associated with cognition
- SZ has a strong genetic component (50% risk in MZ twins). Perhaps genes determining the levels of NMDA receptor function in humans
142
What are 3 genetic factors of NMDA receptor function that may be involved in SZ?
- Lower NR1 (glycine binding site subunit) so fewer funcional NMDA-Rs
- Increased D-amino acid oxidase (DAAO)- increases the breakdown of D-serine
- Increased neuregulin interacts with ErB4 receptor in the postsynaptic density which then downregulates NMDA-Rs via PSD95
143
What happen when you produce a knockdown NR1 mouse?
- Expresses only 5% the normal levels of the NR1 subunit as complete KO is fatal
- Insensitive to PCP (suggests little functionality associated with NMDA-Rs)
- Display symptoms associated with SZ (social withdrawal or isolation and increased locomotor activity and stereotypy)
- Mohn AR et al 1999
144
What happens when you give a knockdown NR1 mouse antipsychotics such as haloperidol and clozapine?
Responds well, potentially target the dopamine system and help mediate positive symptoms
145
Why is it not a smart idea to directly target L-Glu binding site to treat SZ?
Potential for excitotoxicity
Therefore Could target the glycine agonist site to potentiate the NMDA receptor
146
How do NMDA-Rs function?
As heteromers
147
How do we know that NMDA-Rs only function as heteromers?
METHOD
- Inject receptor mRNAs into Xenopus oocytes
- Record whole currents to 100micrometers NMDA and/or 10micrometers glycine agonist applications
RESULT
- GluN1 functional- weak response
- GluN2 non-functional (not trafficked to membrane)
- GluN1 + GluN2- full functionality (Gly agonist binding site 1 and L-Glu is 2)
- Kutsuwada et al 1992, Ishii et al 1993
148
What molecules produce the same effect as glycine?
- Small D amino acids
- D-serine
- D-alanine
- D-cycloserine
(have been evaluated in clinical trials for negative symptoms of SZ)
149
How can we increase levels of D-serine extracellularly for the treatment of SZ?
Inhibit DAAO (breaks it down)
150
How can we increase extracellular glycine for the treatment of SZ?
- Glycine level heavily mainting by astrocytes (Johnson and Ascher 1987)
- GlyT1 reuptakes glycine into the astrocyte
- If interfere with the transport there will be better activation of NMDA-R as more glycine available
151
What were the results of targetting the glycine site to treat SZ?
Positive results, High dose glycine (60g/day) in treatment resistant SZ or a lower dose (30g/day) with antipsychotics- improved negative symptoms (Heresco-Levy et al 1999; 2004)
152
What were the results of targetting the D-serine site to treat SZ?
D-serine (2-8g/day) was equally effective as glycine (Tsai et al 1998; Heresco-Levy et al 2005)
153
What happens when targetting the glycine site to treat SZ when clozapine is added in conjunct?
- No improvement (Evins et al 2000)
- Clozapine increases glycine and D-serine serum levels but might occlude effects of taking on board high levels of amino acid
- Perhaps because clozapine is already elevating glycine and D-serine levels
154
What are 3 new glycine NMDA-R tests that have occured and what did they aim to do?
- Tested on glycine precursors and transporter (GlyT) inhibitors to increase endogenous glycine levels
- Sacrosine- precursor in glycine synthesis (Tsai et al 2004)- no effects
- Bitopertin (RG1678)- GlyT inhibitor (Roche)- clinical trial phase 3 no better than placebo (Kingwell 2014)
- Iclepertin (BI425809)- More potent GlyT inhibitor, which is effective in improving cognitive function in SZ patients (Fleischhacker et al 2021)
155
What is a potential endogenous psychotogen causing SZ?
Kynurenic acid (KYNA) which is a selective antagonist for NMDA-Rs over AMPA-Rs or KA-Rs and alpha 7-nAChRs at low microM
156
What does endogenous KYNA do?
Mimics acute effects of PCP on VTA (DA) neurons in animals in vivio- increases firing (Erhardt and Enberg 2002)
157
Why is KYNA thought to play a role in SZ?
- 40-50% increase of it in the CSF of SZ patients over control subjects (Linderholm et al 2012)
- Is stress induced, levels are greater in distress-intolerant SZ patients who have heightened sensitivity to stress (Chiappelli et al 2014)
- Lowered levels of KYNA in rats and monkeys improves cognitive function in spatial learning and attentional WM tasks (Kozak et al 2014)
158
Why is KYNA an effective drug pathway to try and treat?
- Clear synthesis pathway which is druggable
- Target Kynurenine aminotransferase to prevent the production of KYNA
159
What percentage of the population suffer from major depression?
15-20%
160
What is the leading antidepressent theory?
- Monoaminergic (5HT, NA) therapy which has changed little over 50 years
- Therapeutuc principle is to increase monoamine levels in the CNS
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How does the monoamine theory of depression increase monoamine levels in CNS?
- Reuptake inhibitors (SNRIs, SSRIs)
- Monoamine oxidase inhibitors (MAOIs)
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What are three major limitations with the monoamine theory for treating depression?
- Latency or lag in effectiveness of at least 2-4 weeks, remission >7 weeks, suicide risk during this period, 15% and problems with compliance
- <50% of patients ever become symptom free
- 33% of patients exhibit monoamine treatment-restistant depression (TRD)
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What happens when ketamine is giving as IV at a sub-anaestetic dose (0.5mg) to depression sufferers?
- Rapid reduction in depression in treatment-resistant patients
- Effect within 2 hours, last for over 1 week
- No lag, single dose, long lasting effect
- Zarate et al 2006
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What is ketamines chemical makeup?
A racemate of two isomers:
- Arketamine
- Esketamine
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Which one of ketamine's two isomers is has been approved as a treatment for depression?
- Esketamine- the more potent of the two
- Approved as a nasal spray Spravato for use in TRD (2019)
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What does post-mortem imaging show about a depressed brain?
- Lower brain volume
- Neurons smaller in size and lower density
- loss of dendritic spines (gray matter) in dorsolateral PFC
- Kang et al 2012
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What is the dorsolateral PFC responsible for?
'Stress target' in order to identify strategies to get over the stress
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What do animal models of chronic stress show in the brain?
- Reduction in dendritic branch length
- Reduction in spine numbers in rat medial PFC (analogous with the human DLPFC)
- Liu & Aghajanian 2008
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What are 7 ways to cause chronic stress in rodents?
- Repeated exposure to cold temps
- Lights on at night
- Wet bedding
- Restraint
- Isolation
- Maternal deprivation
- Repeated administration of corticosterone
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What does imaging in DLPFC in patients with major depression show?
- Decreased AMPA-R subunits GLuA2 and GluA3 (Beneyto et al 2006)
- Increased extracellular glutamate (Hashimto et al 2007)
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What do scans of rodents brain in chronic stress models show?
- Decreased AMPA-R and NMDA-R mediated synaptic responses (EPSCs) and receptor subunit expression (Yuen et al 2012)
- Increased inhibition which decreases excitation and synaptic L-Glu release (McKlveen et al 2016)
- Decreased L-Glu uptake so increased extracellular glutamate (de Vasconcellos-Bittencourt et al)
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What does a rat prefrontal cortex layer V pyamidal cells look like following an acute dose of ketamine?
Originally decreased numbers of spines following 21 days of chronic unpredictable stress (CUS)
But increased numbers of spines to control levels after 1 day if subsequently treated with acute ketamine on day 21 CUS
Li et al 2011
Also found that head diameters on tufted dendrites increase after 24 hours
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What is acute unpredictable stress?
Using a random set of different or unpredictable stressors
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What does the improvement in rodent symptoms suggest after an acute dose of ketamine?
Suggests that there is glutamate receptor trafficking and possible protein synthesis to support synaptogenesis
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What is the forced swim test (FST)?
- Mice are placed on the surface of water and the time they spend immobile is measured
- Anti-depressant effect is when there is reduced immobility
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What is learned helplessness (LH)?
- Initially receive ekectric shocks in a chamber from which they cannot escape
- After training, an escape route is provided to a safe adjacent chamber
- They are unlikely to use this escape route when shocked again when they exhibit LH
- Anti-depressant effect is when there is an increased number and speed of escapes to the safe chamber
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How does ketamine effect animal behaviour in FST and LH?
Antidepressent effects still present up to 2 weeks after administration (Maeng et al 2008; Autry et al 2011)
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What happened when a rodent was given MK-801 compared ketamine in CUS and FST?
- Deletion of NR2B subunit occluded ketamine antidepressant action in FST
- NR2B subunit containing NMDA-Rs?)
- Longer time course of antidepressant effects of ketamine over MK-801
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What blocked ketamines antidepressent effect in the FST?
- NBQX
- Mediated downstream of NMDA-R block by activation of AMPA/KA receptors
- Blocks AMPA but does not cause a huge effect in terms of immobilising individual that receives it
- (Maeng et al 2008)
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What does ketamine increase at the molecular level when given an antidepressent dose?
- Increases phosphorylation and levels of GluA1 subunit
- Increases AMPA-R trafficking and insertion postsynaptically
- Increses phosphorylation of rapamycin (mTOR) which increases RNA/protein translation (protein synthesis)
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What is rapmycin (mTOR)
Serine/threonine protein kinase
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What effects on ketamine does mTOR have?
- Ketamine and Ro25-691 antidepressant effects blocked by mTOR
- Ketamine-induced increase of GluA1 subunit expression blocked by mTOR
- Ketamine-induced synaptogenesis blocked by mTOR
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What is the effect of ketamine in KO BDNF and knock in impaired BDNF trafficking?
- Antidepressant effect is reduced or absent in BDNF trafficking impaired knock-in mice (Liu et al 2012) and KO mice (Autry et al 2011)
- Synaptogenesis impaired in BDNF trafficking impaired knock in mice
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What happens to BDNF and eEF2 levels when ketamine and MK-801 are administered at antidepressant effect doses?
- BDNF protein levels rise
- Dephosphorylation of eEF2 protein which causes its activation (by preventing NMDA-R activation of eEF2 kinase?)
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What happens when eEF2 kinase is blocked?
Increase in BDNF protein levels
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What is BDNF?
Brain-derived neurotrophic factor
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What is eEF2 and what is it responsible for?
- Eukaryotic elongation factor 2
- Promotes amino acid chain elongation during ribosomal translation/synthesis
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Summarise the mechanism of ketamine
- Increases GluA1 subunit and insertion of AMPA-Rs supports behavioural antidepressant action and associated synaptogenesis?
- Triggered by a burst of glutamate release and initial AMPA-R activation?
- Increase phosphorylation of mTOR- mediated by the release of BDNF/TrkB receptor signalling
- Increased production of GluA1 subunit and insertion of AMPA-Rs?
- eEF2 increases and increased translation of BDNF
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What actions do monoamine reuptake inhibitor antidepressants do that parralell ketamine?
- Increase BDNF
- Increase AMPA-Rs
- Induce synaptogenesis
- But these occur over longer time scales as they require chronic administration
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What is the mechanism for disinhibition in which ketamines actions are explained?
- Decreased activation of GABAergic interneurons (NMDA-Rs)- Inhibit the inhibtor
- Decreased inhibition of pyramidal neurons
- Brief increase in L-Glu release
- Increased synaptic AMPA-R activation
- BDNF release
- TrkB activation
- Increased mTOR activation
- Increased AMPA-R insertion
- Synaptogenesis
- (Aghajanian, Duman group)
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What are the two mechanisms attempting to explain ketamines actions?
- Disinhibition
- Direct postsynaptic action
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What is the mechanism for direct postsynaptic action in which ketamines actions are explained
- Decreased tonic activation of postsynaptic extrasynaptic (NR2B) NMDA-R signalling
- Decrease in inhibition of eEF2
- Increase in BDNF
- Synaptogenesis
- (Lacks the involvement of mTOR)
- (Monteggia, Kavalalli group)
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What is a new suggestion about ketamine metabolites and their effect?
- NMDA-R inhibition-independent antidepressant actions of ketamine metabolites
- In mice, a metabolite (2R, 6R-HNK) of Arketamine produced an antidepressant effect in behavioural models of depression and caused underlying synaptogenesis
- Zanos et al 2016
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What is a further finding of 2R,6R-HNK which suggests the blockage of NMDA-R may not be necessary?
- 2R, 6R-HNK was inactive at NMDA-Rs
- The antidepressant action was independent of mTOR activation
- (Interestingly, the antidepressant action of ketamine also appeared independent of mTOR activation in this study)
- Zanos et al 2016
