NTProfile Flashcards
NT types
1) Amino acid neurotransmitter (-COOH)
e. g. Glutamate, GABA, Glycine
2) Amine neurotransmitters (N)
e. g. Ach, Norephinephrine, Dopamine
3) Peptide neurotransmitters (big)
e. g. Substance P, ligand for opioid receptors
Example NT for AA Amines Purines Peptides FA
AA: GABA, Glutamate, Glycine Amines: Ach, DA, Epinephrine, Histamine, NE, 5HT Purines: ATP, Adenosine, GTP Peptides: Substance P, Pain related etc FA: cannabinoid
Technique: How can NT be labelled
- Inject NT candidate
2. Withdraw specific antibodies (Marked with a label)
Antibodies can be raised against
- Specific synthesizing enzymes like GAD65 and 67 for GABA
- The vesicular transporters -> good target as it is relatively big in size
- The high affinity reuptake transporters
- Small neurotransmitters like GABA or Glu themselves -> by beads labelling but it is a little unspecific
Why is identifying type of NT important?
To classify type of neurons
Other (less common) way for labelling NT
1) In situ hybridisation
Radioactively labelled probe with the complementary nucleic acids binds to mRNA of the target
2) Genetic manipulation
Use of GFP either cloned into a NT gene
Only way to experiment while alive allowing it to conduct some electrophysiology
Where are NT synthesised?
Generally, small neurotransmitter are synthesized in the synapse whereas peptide neurotransmitters are synthesized in the soma!
Peptide neurotransmitters:
- Are synthesized in the soma as precursor proteins
- Often one mRNA codes many repetitions of the same molecule
- The precursor proteins are processed in the vesicle while being transported to the axon
How do positively charged NT get stored?
e.g. monoamines, Ach
Exchange: 2H/NT
1. Acidification of vesicle via vATPaseRaising both chemical (pH) and electrical (+) gradient by pumping in H+ using ATP
2. The extra proton provides E for pumping in the NT
3. For positively charged, !electrical gradient! allows the exchange of proton and NT+. Which leads even more acidic in vesicle, leading to exchange of H+ and Cl-
How do negatively charged NT get stored?
e.g. Glutamate
Exchange: 1H/NT
1. Acidification of vesicle via vATPaseRaising both chemical (pH) and electrical (+) gradient by pumping in H+ using ATP
2. The extra proton provides E for pumping in the NT
3. For negatively charged, the !electrical gradient! allows the exchange of proton and NT-
Which NT is neither positive or negatively charged?
GABA
What does a sudden increase in error bars mean?
In general, when a research shows a graph which includes a sudden increase/difference in error bars, it could be explained by change in sample size (some died etc)
Q. What is the effect of acidification during vesicle loading?
Assist to pack the NT molecule more densely, by making it more neutral
Q. Is the synaptic cleft also acidified? What are the consequences?
Yes, it changes the neurotransmission!
e.g. 1) @ retina
In the dark, Na+ channels are constantly open leading to “dark” current -> increase in vesicle release
e.g. 2) @ photoreceptor (retina)
Acidification of cleft suppressed the current
e.g. 3) @hippocampus
ASIC1a currents are depolarizing and activate inhibitory interneurons
How is acidification of synaptic cleft related to epilepsy?
Acidosis evoked EEG seizures were NOT weakened for mice deficient in ASIC1a (pH sensitive cation channel). pH drop mediates a cationic current, leading to depol of neurons. This allows inhibition of epileptic symptoms as
1) depol neurons are inhibitory (@ hippo: inhib > exit, opposite of amyg)
2) Inhibitory interneurons have larger acidosis induced currents than pyramidal cells
3) Interneurons in wt respond to acidosis with increased activity
=> ASIC1a currents are depolarising and activate inhibitory interneurons
What are the players for controlling acidification in neurotransmission
Not only proton but also chloride
What drugs are used to regulate NT transport/loading into vesicles?
1) Reserpine inhibits vesicular monoamine transport and leads to MA depletion, leading to clinical depression
2) Amphetamines lead to the release of vesicular MA stores into cytoplasm and synapse are psychostimulants and can induce psychosis
What are the general steps for neurotransmission?
1) precursors travel along the axon
2) precursor get altered to NT around axon terminal
3) NT get loaded into vesicle via vesicular transporter
4) release
5) receptor action
6) inactivation (reuptake, glia, diffusion or degradation)
What are the example of inactivation
- degradation of the Neurotransmitter (ACh)
- by reuptake (Glu, Gaba) and passively by diffusion
Glu(tamate)/Glutamic acid Profile
!Source/metabolic origin: Kreb cycle or glia
Precursor: glutamine α-Ketoglutarate
!Enzyme: PAG & Transaminase
!Storage/Transporter name: vGluT
E provider: electrochemical (& H+ antiport)
!Inactivation/Transporter name: no enzymatic/EAAT
E provider: Na+ gradient
Localisation: Glia and neuron
!Recycling: Reuptake and glial Gln Shuttle
Glutamate synthesis & enzyme()
1) α-Ketoglutarate (mostly from KREB, some from glia)
- (Glu-aminotransferase/aka transaminase)-> Glu
2) Glutamine
- (Phosphate Activated Glutaminase)->Glu
Glutamate Storage
- via vGluT
- Highly specific; charged by ELECTROchemical gradient of proton
- 3 isoforms (1&2 neuronal, 3 neuronal & astrocyte)
- also act as a phosphate carrier/transporter, on presynaptic cell, which activate PAG to synthesise more Glu
Glutamate inactivation
- via Exitatory AA Transporter
- E used for plasma membrane: Na+
- E used for vesicle memb: proton and ATP
- co-transport of K+, which restores the original configuration of the transporter (Glu binding site open to the outside)
- Cl-conductance is independent of Glu binding and transport
- 2 modes of operation:1) transient and fast buffering in which the availability of Glu is reduced but no real transport takes place (just binding)2) actual transport of Glu molecules
EAAT localisation
Inactivation transporter for Glu
@hippo
-EAAT1&2 on glia
-EAAT3&4 on neurons
-Glia cells in this region only marginally/loosely surround synapses-> allows Glu to accumulate in cleft-> allows Glu to activate nearby cleft
-Findings with TBOA (EAAT inhibitor)
Inhibition of Glu (excitatory AA) uptake seems to affect synaptic transmission only at high frequencies
@ cerebellum
-Glia cells (Bergmann cells) in this region only tightly surround synapses-> efficient uptake
-Findings with TBOA
Inhibition of Glu (excitatory AA) uptake seems to affect synaptic transmission both at low and high frequencies
What is TBOA
EAAT inhibitor
Glutamate receptors
NMDA: Mg blocked
AMPA: important role in LTP
metabotropicGluR
GABA profile
!Source/metabolic origin: α-Ketoglutarate (Kreb cycle)
Precursor: Glutamate
!Enzyme: GAD 65 & 67
!Storage/Transporter name: VIAAT
E provider: H+ antiport & electrochemical
!Inactivation/Transporter name: no enzymatic/GAT
E provider: Na+ gradient
Localisation: Glia & neuron
!Recycling: reuptake
Glutamate synthesis & enzyme()
Storage
Inactivation
Receptor/recycling
!Source/metabolic origin: Precursor: !Enzyme: !Storage/Transporter name: E provider: !Inactivation/Transporter name: E provider: Localisation: !Recycling:
GABA synthesis & enzyme()
de-carboxylating (CO2) glutamate
GABA = Glutamate with COOH removed
Glu
- (glutamic acid decarboxylase (GAD, 2 isoforms GAD 65 and GAD 67))
- > GABA
Where is GABA located?
Only in neural tissue (Glu & Gln is everywhere)
GABA storage
1) vGAT = vesicularGABAtransporter GAT1-3
2) VIAAT = vesicular inhibitory aminoacid transporter (includes vGAT)
- vGAT has a low substrate affinity
- intracellular GABA content is high
- GAD-65 associates directly with synaptic vesicles.
- VIAAT also works for glycine (AA)
VIAAT knockout
VIAAT: transporter for GABA&Glycine vesicle loading
KO reduce amplitude and frequency of IPSC
Why does VIAAT depend on equal amount of electric and pH gradient?
VIAAT: transporter for GABA&Glycine vesicle loading
Because movement of 1 GABA molecule equal shift in 1 proton & 1 positive charge at the same time
GABA inactivation
- High affinity GAba Transporter bring in 2 Na, 1 Cl & 1 GABA
- GAT located both on neurons and glia
Drug used for GABA inactivation
- Tiagabine as antagonist for GAT has antiepileptic and anxiolytic effects
- Nipecotic acid
GABA receptor (not on test)
Blockade of GABAAR (ionotropic) reveals large tonic currents in GAT-1 knockout => GAT-1 regulates tonic GABAAR mediated inhibition
Drugs to block GABA A receptor
benzodiazepines, barbiturates, alcohol
Breakdown of GABA
- GABA can breakdown into so many molecules such as Gamma HydroxyHutyrate
- While GHB is naturally derived, making it hard to detect when taken as drug
- It can be externally taken as drug, GABAB-receptor agonist
- Medical use: Xyrem, narcotic (Norway), Cataplexy, daytime sleepiness in narcolepsy (USA)
- “Date rape drug” (aka „fantasy“, „liquid X“ or „liquid extasy“)
- Low concentrations: euphoria, impaired judgement, anxiolysis and amnesia
- High concentrations: unconsciousness, seizures, respiratory failures, coma, death
GHB acidura
- Elevated GHB levels in urine, plasma and cerebrospinal fluid
- Cause Mental and motor retardation, ataxia
- Due to SSADH deficiency (oxidizes Succinic SemiAlDeHyde to succinate)
Glycine Profile
!Source/metabolic origin: Serine Precursor: - !Enzyme: Serin hydroxymethyltransferase @mitochondria !Storage/Transporter name: - E provider: - !Inactivation/Transporter name: GLYT1 and GLYT2 E provider: - Localisation:- !Recycling: -
Ach Profile
!Source/metabolic origin: Blood (choline), Acetyl CoA
Precursor: (Phosphadityl) choline between kreb cycle and glycolysis
!Enzyme: ChAT
!Storage/Transporter name: VAT
E provider: H+ antiport
!Inactivation/Transporter name: enzymatic inactivation by Ach esterase/ChT
E provider: Na+ gradient
Localisation: Neuron
!Recycling: Only Choline reuptake
Ach synthesis & enzyme()
originate from 3 nuclei (brainstem nuclei, nucleus basalis of Meynert & medial septal nucleus and diagonal band)
Phosphatidylethanolamine (memb protein) -------> choline + Acetyl CoA -(choline acetyltransferase)-> Acetylcholine -(acetylcholinesterase)-> choline & acetate
Recycle because choline get reused and acetate + coenzyme = Acetyl CoA
Ach characteristic
-Not CNS NT; locally located & low in number while projecting to the whole brain (except cerebellum)
Ach Storage
- via VAchT
- VAchT closely related to VMAT
- low binding affinity and transportation velocity because of high intracellular Ach concentration (VMAT have high binding affinity because of substrate cytocoxicity)
Which has enzymatic deactivation?
Purines (ATP) and Ach
Ach Recycling
Phosphatidylethanolamine (memb protein) -------> choline + Acetyl CoA -(choline acetyltransferase)-> Acetylcholine -(acetylcholinesterase)-> choline & acetate
Recycle because choline get reused (Na dependent active transport system) and acetate + coenzyme = Acetyl CoA
Ach Inactivation
1) enzymatic via Ach esterase
2) reuptake of Choline via ChT
- ChT are high affinity
- localised on pre synapse
- Na+, (Cl-) gradient dependent transportation process
- Choline uptake is rate limiting step for Ach synthesis
- ChT is already present on NT vesicles
Drug for ChT
Choline reuptake transporter
-HC3 is a specific ChT blocker and interfere with learning
Drugs for Ach Nervous System
Physostigmine: Inhibits Achesterase
Sarin: Inhibits Achesterase; Increase Ach, tonic arrest of muscle
HemiCholinium3: Block Choline uptake
Muscarine: GPCR
Nicotine: Ionotropic R first discovered
Vesamicol: Blocks Ach transport into vesicle
Where is Acetyl CoA produced?
In the mitochondria
From where is choline obtained?
From blood phosphditylcholine; choline is rate limiting factor for Ach synthesis
Other potential/unsolved source for Ach synthesis
glucose, citrate, acetate
More drugs for Ach
Vesamicol: inhibit the vesicular AchT
Botulinus & tetanus: inhibit Ach release
Black widow spider toxin: stimulate Ach release
Curare: nitotinic antagonist
Atropine: muscarinic antagonist (eye drop)
AchR
nAchR, GPCR (M2,4 or M1,3,5)
M2 both on pre and post
Catecholamines
Amine group (N) + benzene + 2 OH e.g. Dopamine, Norepinephrine, epinephrine
Catecholamines & serotonin
- few neurons which project widely like Ach
- almost exclusively GPCR (except 5HT3R)
- not dedicated to fast and precise information transfer but mediate global state (attention, emotions, arousal) just like Ach
- classically termed “neuromodulators”
Enzymes used to label DA for imaging
TH & DBH
Dopaminergic produced at
1) arcuate nucleus
2) substantia nigra
3) ventral tagmental area
Dopaminergic projections
1) Hypothalamus (arcuate nucleus -> hypothalamus, anterior pituitary)
2) Mesostriatal/nigrostiatal pathway (substantia nigra -> dorsal striatum)
Diminished in PD
3) VTA (Ventral striatum (NA); olfactory bulb, amygdala, hippocampus, medial/orbital prefrontal cortex, cingulate gyrus)
Related to reward/addiction
DA receptor projection
D1-D5: celebral cortex & limbic system
D3&D5: hypothalamus
D1&D2: corpus striatum
Function of DA receptor
D1 & D5 activation of adenylate cyclase (cAMP)
D2-D4 inhibition of cAMP
Noradrenaline/norepinephrine projection
Locus ceruleus:
- 50% of all NA producing neuron lie here (others are from brainstem A1-A7)
- Just ~12.000 neurons in the human
- Important in sleep/wake cycle and arousal regulation
β-adrenerigc receptors
- Fear engrams
- Fear recall
- PTSD
!!!Biosynthesis for catecholamines
@pre-synaptic L tyrosine -(Tyrosine Hydroxylase)-> L-DOPA -(DOPA decarbohyxylase/L-aromatic AA DeCarboxylase/L-AADC)-> Dopamine @axon terminal -(Dopamine b-hydroxylase)-> Norepinephrine -(Phenylethanolamine N methiltransferase) -> epinephrine
L-DOPA
drug given commonly to PD
Excessive L-DOPA can cause hallucination etc
Tyrosine Hydroxylase
- rate limiting step for catecholamines
- highly regulated enzymes, meaning it has many phosphorylation sites, receptor tyrosine kinase etc
- End product inhibition: Catecholamines compete with BH4, allowing it to prevent catecholamines to be overproduced
Catecholamine Storage
VMAT: Vesicular Monoamine Transporter
- Mg2+dependent
- Reserpine – given to PD
- MPTP/MPP.(Methyl-phenyl-tetrahydropyridine) – kill DA neurons
Catecholamine Inactivation
1) Re-uptake via
DAT: Dopamine Transporter
NET: Norepinephrine Transporter
Energy & Na+ gradient dependent; presynaptic
2) Enzymatic breakdown via Monoamine oxidase (MAO) & Catechol O-methyltransferase (COMT)
-> VMA and HVA (breakdown of DA) are plasma markers for CA metabolism
Drug for catecholamine
Reserpine: inhibit VMAT
Cocaine: inhibit DAT
Amphetamine: increase dopamine release via the DAT
Antipsychotics: block D2 receptors
Animal model of PD
TH KO
VMAT KO
DAT KO
Drugs for PD
MPTP/MPP+
Serotonin projection
Location
Several brainstem nuclei, 5HT neurons are only a fraction in each!
~400.000 neurons?
invade almost the complete brain (including cerebellum)
Function
Sleep, arousal, attention
emotions and mood
Serotonin Synthesis
Tryptophan -(Tryptophan hydroxylase)-> 5-Hydrocytryptophan -(L-aromatic AA decarboxylase)-> serotonin -> melatonin
Serotonin storage
VMAT: Vesicular Monoamine Transporter (exact same as catecholamine)
-Mg2+dependent
-Reserpine – given to PD
-MPTP/MPP.(Methyl-phenyl-tetrahydropyridine)
– kill DA neurons
Serotonin drugs
Reserpine: inhibit VMA; used as antipsychotic (herbal)
SSRI/SNRI: antidepressants inhibit serotoin transporter
Antipsychotics: inhibit 5HTR
MAOI/monoamine oxidase inhibitors: antidepressants inhibit MAO
Serotonin R can be targets for antimigraine
Monoamine (catecholamine & Setoronin)
MA & 5HT constitute “Widely projecting Systems”-> As it is slow synapse, the staying duration of NT can be controlled. This cannot be done for fast synapse e.g. Ach as for example will cause complete relaxation of muscle
Small groups of neurons innervate large areas of the brain
Diffuse targets
No information processing, but rather general mental and physiological states and mood
Important for many diseases
