NT-synthesis II- Salmen Flashcards
characteristics of NTs
- must be synthesised and released by the neuron
- should reproduce the effect of presynaptic nerve stim. when applied exogenously
- dose dependent blockade of its action w. competitive antagonist should be possible
- blockade of synthesis should block the effect of presynaptic synthesis
- NT-action should be terminated primarily by active mechanisms: inactivation, reuptake, but also passively by diffusion
classes of NTs
- Amino acids - GABA, glycin, glutamate
- Amines: Dopamine, 5HT, ACh, Norepinephrine, histamine
- Purines: ATP, GTP, adenosine
- Peptides: Dynorphin, enkephalin, NAAG, NPY, substance P
- Fatty acids: Endocannabonoids- anandamide, 2AG, THC
how can one use NT candidate and antibodies to label neurons? (process)
- inject NT candidate
- withdraw specific antibodies
- brain tissue section –> labeled neurons containing NT candidates
- NT candidates are attached to AB that are chemically tagged with a visible marker
what can one raise AB for?
- specific synthesising enzymes like GAP65 for GABA
- specific vesicular transporters
- the high affinity reuptake transporters
- also against small NTs like GABA and Glu
how can one label neurons according to their NTs using in-situ hybridisation?
- inject strand of mRNA into neuron
- radioactively labeled probe with proper sequence of complementary NA
- brain tissue section
how can one label neurons and NTs using brain manipulation?
- use GFP either cloned into NT gene (e.g GAD 67)
- one will receive GAD67-GFP mice (labeled for GABA)
- GABAergic neurons will be fluorescent
which of the following answers is WRONG?
a. generally small NTs are synthesised in the synapse directly whereas peptide NTs are synthesised in the soma
b. peptide NT are synthesised in the soma as precursor proteins, which are then being processed in vesicles and transported to axon.
c. peptide NT are synthesised in the synapse whereas small NTs are synthesised in the soma and transported to the axon via vesicular transporters
d. often 1 mRNA molecule codes many repetitions of the same NT molecule
c
how are NTs loaded into vesicles?
- NTs are acidifying the vesicles
- transport of 1 proton provides the vesicle with energy
- energy driven ATP –> the energy comes from a build up of pH gradient generated by V-ATPases
- gradient of molecules + electronic gradient (positive charge inside the vesicle)
what are the effects of acidification? is the synaptic cleft also acidified?
- packing NT into vesicle requires energy which comes from a build up of pH grad. generated by V-ATPases
- in most cases, the pH is adjusted tp keep the NT molecules electrically neutral, to enable higher density of NT molecules in the vesicle
what are the effects of acidification after release in the retina?
- in the dark, Na+ channels are always open –> dark current
- dark current is maintained by cGMP
- the depolarising dark current leads to SV release
- light triggers conformational change in rhodopsin –> hydrolysis of cGMP to GMP –> Na+ channel closes –> dark current disappears –> cell repolarises, and SV release stops.
- Problem: adaptation of the system to different light intensities.
what are the effects of acidification after release during hippocampal seizures?
- Evoked EEG seizures attenuate after some time in WT but not in ASIC1a-KO mice.
- In-vitro seizure generation is suppressed by acidosis in WT but not in KO
what is ASIC1a + function?
- a drop in pH leads to a cationic current –>
depolarisation of neurons. - How can this terminate epileptic seizures? –> inhibitory neurons have larger acidosis-induced currents than pyramidal cells–> ASIC1a currents are depolarising & activate inhibitory neurons.
Which statement is CORRECT?
A. Monoamines, ACh and GABA depend predominantly on pH gradient for vesicle loading whereas glutamate depends mostly on electrochemical gradient for vesicle loading
B. Monoamines and glutamate relay mainly on pH gradient whereas GABA depends mostly on electrochemical gradient
C. For vesicle loading, Monoamines and ACh are driven only by pH gradient, GABA depends mostly on pH gradient and slightly on electrochemical gradient, and Glutamate’s loading is driven predominantly by electrochemical gradient
D. Vesicle loading of all neurotransmitters is pH gradient-driven
C
Give 2 examples for drugs that block NT loading into vesicle
- Reserpine: inhibits vesicular MA transport —> MA depletion—> clinical depression
- Amphetamine: release of vesicular MA stores into cytoplasm and synapses—> psychostimulants—> can induce paychosis
What are the key enzymes in the synthesis of glutamate?
- Glu-aminotransferase - converts alpha ketoglutarate (from Krebs cycle or from glia) to glutamate
- Phosphate-activated-glutaminase (PAG)- converts glutamine into glutamate. —> Neurons are unable to synthesise glutamine bc they are lacking Gln-synthetase, so same as alpha ketoglutarate it is released by glia and taken up by neurons
