Epilepsy Flashcards
What is epilepsy?
A disorder of the CNS characterized by recurrent, sudden, large increases in electrical activity (electrical seizures) that may be localized or generalized
What determines Neuronal output (fire action potential or not)?
– Determined by number of excitatory and inhibitory synaptic inputs – Strength of individual inputs
- Integrated response
What can synapse transmitters do?
• Synaptic transmitters can:
– Excite neurons – depolarize
- Inhibit neurons – ‘hyperpolarize
• Also can modulate activity ion channels involved in: – Transmitter release (voltage gated calcium channels)
– Controlling membrane excitability (potassium channels)
• Changes frequency/number of action potentials
What is the role of glutamate in the CNS?
- Glutamate – major excitatory transmitter in CNS
- Mediates most of fast excitatory neurotransmission
– ~70-90% of CNS synapses - glutamatergic
• Principle mediator of sensory information, motor coordination, emotions, cognition (including memory)
Where does glutamate act?
Acts on specific receptors – Ionotropic receptors (ion channels)
Metabotropic receptors (G protein coupled receptors)
What does aspartate do in the CNS?
– mediates transmission at a small number of central synapses
How is the brains glutamate supply maintained?
– Non-essential amino acids – Do not cross blood-brain barrier not supplied by circulation
– Synthesized in brain from metabolism of glucose
- Also from glutamine synthesized by astrocytes
What happens to glutamate after it has been released into the synapse?
Released glutamate taken up primarily by astrocytes
Glutamine Synthase – Converted into glutamine by glutamine synthase
– Glutamine transported out of astrocytes
– Glutamine uptake by neurons (transporter)
– Converted back to glutamate by the enzyme, glutaminase
Which molecule of the TCA cycle is converted into glutamate?
Alpha-Ketoglutarate
How do synaptic vesicles accumulate glutamate?
– electrical gradient created by different concentrations of H+
across vesicle membrane (i.e. inside vesicle and in cytoplasm)
• Vesicle positive potential with respect to cytoplasm
– Electrical potential gradient generated by vesicle ATP proton (H+) pump
What is the synaptic vesicular concentration of glutamate?
• Vesicle concentration >20mM
– 1,000 – 2,000 molecules per vesicle
Describe the sequence of events of Release and reuptake of synaptic glutamate
• Action potential in pre-synaptic neuron
– Depolarizes the pre-synaptic terminal
– Depolarization opens voltage gated calcium channels
– Calcium flows into terminal
– High local concentration of intracellular Ca2+
– Triggers exocytosis of synaptic vesicle contents ok
• Glutamate diffuses across synaptic cleft
– Interacts with specific receptors
– Multiple receptor types
• Reuptake of glutamate (and aspartate)
– Excitatory Amino Acid Transporters (EAATs)
– Reduces the extracellular concentration
– Terminates transmitter action
How does glutamate reuptake work?
• Glutamate transporters drive uptake through EAAT
– Co-transport of (2-3) Na+ and H+ into the cell
– Counter-transport of K+
• Most transporters located on
– Glial cells (astrocytes)
– Post-synaptic neurons (lesser extent)
Post-synaptic Exocytosis
• Some glutamate may diffuse to act at adjacent synapses
(• Km for glutamate ~low micromolar
– Keeps extracellular concentration low
• ~15,000 – 20,000 transporters per synaptic bouton –
Effective uptake process)
Outline the different types of glutamate receptors?
• Two large families of receptors
• Ionotropic receptors
– ion channels Ionotropic
– Binding site located on channel
– Agonist binding promotes channel opening
– Role in fast synaptic transmission
– 3 classes:
• NMDA (N-methyl-D-aspartate) receptors AMPA
• AMPA (-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors
• Kainate receptors (kainate found in some seaweeds)
• Names based on ability of these drugs to selectively activate channels
• Glutamate is the natural transmitter at all receptors
• Metabotropic receptors
– G protein coupled receptors
– Modulatory effects on neuronal function and synaptic transmission
See page 12 of glutamate excitatory lecture for a table?
-
Three agonists that work at AMPA receptors?
An antagonist?
Glutamate
AMPA
Kainate
CNQX
Three agonists that work at NMDA receptors?
An antagonist?
Glutamate
Aspartate
NMDA
D-AP5, D-APV, MK-801
Ketamine
Phencyclidine
How are glutamate receptors distributed in the brain?
• AMPA & NMDA receptors often co-localise at functional excitatory synapses
– Ratios at individual synapses varies greatly
– Some can contain only one sub-type
– Some can contain only one sub-type
• Only small number of kainate receptors in most CNS regions
What is the structure of ionotropic glutamate receptors?
- Each subunit has 3 transmembrane spanning domains
- Large extracellular N-terminus
- Receptors made up of 4 subunits
What type of excitatory transmission are AMPA receptors involved in?
• AMPA receptors
- fast excitatory synaptic transmission
– Fast synaptic current
– Fast decay due to relatively low affinity (Kd ~200nM)
What type of excitatory transmission are NMDA receptors involved in?
• NMDA receptors
Fast excitatory neurotransmission
– Slower onset than AMPA
– Slower decay (up to several hundred msecs) – why?
– Higher affinity glutamate binding (Kd ~ 5nM)
- glutamate Stays on receptor even after synaptic clearance of glutamate - goes between open and closed state
What a AMPA receptors assembled of?
What is the relative synaptic current speed?
To what are they permeable?
- Assembled from GluR1-4 – Tetramers
- Fast synaptic current
• All permeable to Na+ & K+ (some also Ca2+-permeable)
– Depolarizes towards reversal potential, ~0mV
- Activation depolarizes neuron
– Activation depolarizes neuron
To what ions are NMDA receptors permeable?
• NMDA receptors are permeable to Na+, K+ and Ca2+
– Ca influx can also activate 2nd messenger systems and Ca dependent enzymes
What does the slower action of the NMDA receptor allow?
• Slower action of NMDA receptors
– Provides mechanism for spatial & temporal summation
– Also voltage sensitive
• At membrane potentials
What is meant by ‘NMDA receptors act as ‘co-incidence’ detectors (i.e. several inputs)’?
– Needs repetitive or multiple excitatory inputs from other AMPA receptors -depolarize the neuron and relieve the Mg2+ block depolarize the neuron and relieve the Mg block
– Act to sense activity of many independent synaptic inputs on same neurone
What is the structure of an NMDA receptor?
• Tetramers
– 2 NR1 subunits plus 2 NR2 subunits
What factors are require to make an NMDA receptor function?
– Glutamate binds to the NR2 subunit
– Binding of glycine ( or D-serine) to a site on NR1 – (not to be confused with inhibitory glycine receptors)
- Glycine concentration in brain saturating for some sub-types
- Potential site for drugs to act : drugs that prevent glycine binding will inhibit NMDA receptorsa
What drugs block NMDA receptors?
Phencyclidine
Ketamine
Dextromethorphan
What effects do NMDA receptor antagonists have in small concentrations?
• Psychotomimetic
− Cognitive defects
− Hallucinations
− Delusions
What effects do NMDA receptor antagonists have in higher concentrations?
• Dissociative anaesthetics
− Sensory dissociation
− Analgesia
- amnesia
What is the the NMDA antagonist Dextromethorphan used as?
Cough suppressant
What type of drug is ketamine and what is it used for?
NMDA receptor antagonist
Ketamine: anaesthetic/analgesic
-pediatric anaesthesia, emergency surgery, (usually with sedative drug e.g. Diazepam). Suppresses breathing less than most other anaesthetics and increases cardiac output.
How many Metabotropic Glutamate receptors are there?
- 8 receptors known mGluR1 8
* 7 transmembrane G-protein coupled receptors
Are metobotropic glutamate receptors made up of subunits?
• Not formed of subunits – One molecule = one receptor
How are metobotropic glutamate receptors grouped?
• Grouped into 3 groups according to:
– Amino acid sequence homology
– Agonist pharmacology
– Signal transduction pathways
– Group I, II & III
Where are metobotropic glutamate receptors located?
Located in different regions of CNS
– Pre-synaptic
– Post-synaptic
Which metobotropic glutamate receptors are in Group I, and what is the pathway that they activate?
Group I: mGluR1, mGluR5 - Activates Phospholipase C - production of IP3 and diacylglycerol (DAG) • IP3 - Ca2+ release -increase intracellular Ca2+ concentration • DAG activates Protein Kinase C protein phosphorylation Modifies ion channel activity
(Beta-gamma complex dissociated can interact with ion channels directly)
Which metobotropic glutamate receptors are in Groups II and III and what is the pathway they are in?
Group II:
mGluR2, mGluR3
Group III:
mGluR4,
mGluR6-8
- Inhibits adenylate cyclase
- decrease cAMP levels
- Modifies ion channel activity
(Beta-gamma complex dissociated can interact with ion channels directly)
What are the modulators effects of pre-synaptic metobotropic glutamate receptors?
– Inhibit voltage-gated Ca channels – reduce transmitter release • Primarily group II & III receptors
– Glutamatergic terminals (inhibitory autoreceptors)
– Terminals that release other transmitters
What are the modulators effects of post-synaptic metobotropic glutamate receptors?
• Post-synaptic effects - variable
– Inhibit some K+ channels
– increase excitability
– Increase activity of some K+ channels – decrease excitability
– Effects depend on cell type involved
View second to last page of glutamate excitability lecture
-
What do you you know about the link between calcium influx and excitotoxicity?
• Many ionotropic glutamate receptors are permeable calcium – Greatly increased glutamate release seen with a seizure or in ischaemia/reperfusion with e.g. stroke
– Intense receptor activity
• Large Ca2 influx – Overwhelms normal Ca2+ buffering & sequestration into intracellular organelles
- Increased intracellular Ca2+ concentration – Normally used as a signalling pathway e.g. activate some enzymes
- Protein kinase C, Phospholipase C –
BUT large Ca2+ increase
• Activates Ca2+-dependent enzymes that cleave proteins (proteases)
• Generates damaging – Lysophospholipids - compromise membrane integrity – Oxidative stress (free radicals)
• Leads to cell death
(Why they want an NMDA antagonist - Limit the effects of stroke
- would have to be given v. Soon after event though)
What is the major inhibitory neurotransmitter in the brain?
- GABA: gamma-amino butyric acid
- GABA occurs in brain tissues but not in other mammalian tissues at significant concentrations
• GABA functions as major inhibitory transmitter in many CNS pathways
– Functions throughout brain
• Transmitter at ~30% of all synapses in CNS
How is GABA synthesised?
• GABA formed from glutamate by action of enzyme
– Glutamic acid decarboxylase (GAD)
– Enzyme found only in GABA-synthesizing neurons
(• GABA destroyed by GABA Transaminase within cells)
What do you know about vigabatrin?
• Vigabatrin – synthetic analogue of GABA
– Inhibits GABA Transaminase by irreversible covalent binding
– Long lasting effect despite short plasma half-life
Increases GABA concentration in brain
– Increases GABA concentration in brain
– Effective in epileptic patients (resistant to other drugs)
– Generally well tolerated and relatively free from side effects
• Main drawback – depression and occasional psychotic episodes in minority of patients
Ae drugs that increase GABA activity effective in treatment of abscence seizures?
– Absence seizures are paradoxically often exacerbated by drugs that enhance GABA activity
• Better treated by drugs acting by different mechanisms
What does GABA transaminase convert GABA into and what can then happen to this?
Succinic semialdehyde
- can become succinate and enter TCA
How is GABA concentrated in vesicles?
• Vesicular packaging via a transporter –
Like glutamate - transporter dependent on electrical potential across vesicle membrane
• Potential generated by ATP-dependent proton pump
How is GABA taken up from the synapse and by what?
• Released GABA taken up by transporters
– Uses energy from Na+ gradient to drive uptake (co-transport)
– In pre-synaptic neurons : reutilized
– In post-synaptic neurons & glia
What does Tiagabin do?
– inhibits GABA uptake and therefore increases GABA concentration
What are the 2 main functional groups of GABA receptors?
• Ionotropic GABA receptors – post-synaptic
– Site of action of benzodiazepine drugs
• Metabotropic GABAB receptors – pre- & post-synaptic
– G protein coupled receptors
• GABA receptors identified in all regions of the brain
How are GABA receptors structured?
- Each receptor made up 5 subunits
- Each subunits has 4 transmembrane spanning domains
Most likely composition is 2alpha 2Beta 1gamma
Most prevalent combination in brain: alpha1, beta2, gamma2 (60%) Others: alpha2, beta3, gamma2 (15-20%) alpha3, beta3, gamma2 (10%)
GABA binding sites (2) at interface of alpha & beta subunits
Benzodiazepine binding site at interface of alpha & gamma 2 subunits
Expression of each subtype varies in different brain regions (e.g. alpha6 expressed almost exclusively in b ll ) cerebellum)
How do GABA receptors basically work?
• GABAA receptors are Cl- selective ion channels
– Reversal potential near 70mV
– Hyperpolarize neuron or ‘clamp’ voltage near resting potential
• Inhibit depolarization responses to excitatory inputs
Name a GABA receptor agonist?
• Muscimol from hallucinogenic mushrooms
2 GABA antagonists?
• bicuculline
- Convulsant alkaloid - inhibits GABAA receptors by binding to the GABA binding site
• picrotoxin
- Another convulsant - blocks the chloride channel
Types of GABA modulators drugs?
- Benzodiazepines
- Barbiturates
- Neurosteroids (e.g. pregnenelone, alphaxolone)
- Ethanol
- Anaesthetics
How do benzodiazepines work?
Benzodiazepines – Selectively potentiate GABA effects on GABAA receptors
– Bind to an accessory site – not the GABA binding site
- Located at a site of alpha subunit interaction with gamma subunit
- Facilitates binding of GABA by allosteric action
- Enhances effect of GABA Enhances effect of GABA
• Increase probability of channels opening
– no effect on channel conductance or open time
What three GABA agonists of the benzodiazepine family do you know?
What are their relative durations of action?
- Diazepam – long lasting as metabolized to active metabolite
- Clonazepam – long lasting parent compound
- Lorazepam, Temazepam – short lasting, no active metabolite
What is a GABA antagonist, can act as a convulsant and can be used as an antidote to benzodiazepine overdoses?
• Flumazenil
– can act as a convulsant
• Used as antidote for benzodiazepine overdoses
What are the clinical effects of benzodiazepines?
– Anxiolytic
• Used mainly for acute anxiety
• Use reducing in favour of antidepressant drugs and Cognitive Behavioural Therapy.
– Sedative
• Decrease time to sleep onset & increase time asleep
• Sleep effects tend to decline when drug taken for 1-2 weeks –
- Reduction of muscle tone
• Feature of anxiety in humans
• Contributes to aches, pain and headache in anxious patients
• Possible without appreciable loss of coordination
– Anticonvulsant
• Diazepam (i.v.used to treat status epilepticus)
– Acts rapidly compared to other drugs
• Clonazepam claimed to be relatively selective anti-convulsant
– Sedation a concern
– Withdrawal may exacerbate seizures
– Amnesia
• Reduced memory of events experienced while taking Benzodiazepines
• Minor surgery without unpleasant memories
What are the side effects of benzodiazepines during use?
– Drowsiness
– Confusion
– Amnesia
– Impaired coordination
– High degree of tolerance and dependence
What happens in acute toxicity from benzodiazepines?
– Overdose less dangerous than other anxiolytic/hypnotic drugs
– Prolonged sleep without depressing respiration/cardiovascular function
– With alcohol – respiratory depression
• Treated with antagonist, Flumazenil
What are the withdrawal symptoms for benzodiazepines?
– Anxiety, increase in irritability and aggression
– Tremor and dizziness
How might it be possible to separate the undesirable affects of benzodiazepines from the desirable effects?
• Action at alpha1 subunits leads to unwanted side effects (sedation) – Action at alpha2 and/or alpha3 containing receptors responsible for desirable anxiolytic effects without side effects
– Prospect that selective alpha2/3 acting ligands could be superior anxiolytic drugs
What are barbiturates?
• Barbiturates discovered in the early 20th century
• Bind to GABA receptor at different site to benzodiazepines
– Potentiate GABA effect
- Less specific receptor activity than Benzodiazepines
- Widely used as sedatives until 1960s
What can result from barbiturate overdose?
- death from respiratory and cardiovascular depression
Clinical effects of barbiturates?
– Sedative effects
- impaired cognition
- motor coordination
Why are barbiturates rarely used?
– High degree of tolerance and dependence
– Strongly induce hepatic cytochrome P450 enzyme
• Enzymes that break down many drugs
• Drug drug interactions
What three barbiturates do you know and when they are used, what is it for?
• Pentobarbital
- occasionally used for sleeping pills and anxiolytic but less safe than benzodiazepines
- Phenobarbital – still used for anticonvulsant activity
- Thiopental – used as an intravenous anaesthetic
Where are neurosteroids made and when are their levels increased?
- Neurosteroids (e.g. allopregnanolone)
- Locally synthesized in CNS in glial cells from cholesterol or steroidal precursors
• Increased synthesis of endogenous neurosteroids in the brain during
– stress
- pregnancy
- after ethanol consumption
How do neurosteroids affect GABA activity?
• Two binding sites on alpha sub-unit
– Potentiates
– Recent evidence that can also directly activate channel
• Enhance GABA-mediated chloride currents
What synthetic neurosteroid do you know?
• - alphaxolone - developed as anaesthetic agents
– Veterinary usage
Disrupted steroid regulation of GABAergic transmission is implicated in several debilitating conditions. What are these?
- panic disorder
- depression
- schizophrenia
- some forms of epilepsy
How does ethanol affect GABA receptors?
– Multiple sites of action in CNS
– Acute GABA receptor potentiation similar to benzodiazepines
– Longer term modifications (receptor location, subunit composition, neurosteroids etc)
– Other sites include - Voltage gated Ca2+ channels
Do anaesthetics work on GABA receptors?
– GABA receptors one site of action for some general anaesthetics
- Etomidate
- propofol
- thiopental
– Act at different site to benzodiazepines
• Volatile anaesthetics (e.g. isoflurane, enflurane)
He does GABA inhibit nervous transmission presynaptically?
– Inhibition of voltage-gated Ca2+ channels
- decrease transmitter release
– Occurs on glutamate, GABA and other transmitter containing pre-synaptic terminals
He does GABA inhibit nervous transmission presynaptically?
– Increased opening of K+ channels
- reduced firing of post-synaptic neurone
Outline how glycine involved in the CNS as an NT
• A major inhibitory transmitter in vertebrate CNS
– Especially in spinal cord and brainstem
– Critical for regulation of motoneurons
Functions in:
- retina
- auditory system
- sensory systems
What is glycine made from and by what enzyme?
- Serine
- Serine hydroxymethyl transferase
How is glycine stored presynaptically and what takes it up from the synapse?
• Small pool of glycine packaged into synaptic vesicles
– H+-dependent vesicular inhibitory amino acid transporter (VIAAT)
• Released glycine taken up by transporters (GLYT1 & GLYT2)
– Astrocytes – terminates transmitter action
– Pre-synaptic neurons – replenishes pre-synaptic pool
– Driven by Na+ & Cl- gradients
How are glycine receptors structured?
- Like GABAA receptors – pentameric (5 subunits)
- Four types of alpha subunits; one type of beta subunit known
• Composed of alpha (3) & beta
(2) subunits or alpha (2) & beta (3)
– Each 4 transmembrane spanning domains
What compounds that affect the glycine receptors do you know?
- Strychnine (antagonist)
- glycine (agonist)
Both of above bind to alpha subunit
• Picrotoxin – non-competitive inhibitors at glycine receptor alpha subunit
What specifically makes glycine receptors inhibitory?
• Ion channels permeable to Cl-
– Activation generates a hyperpolarizing ipsp
What therapeutic drugs do you know that act on glycine receptors?
• No therapeutic drugs act specifically on glycine receptors
Do you know a condition caused by a mutation in the glycine receptor?
Hyperekplexia
– Rare dominant mutation - single amino acid on alpha1 subunit
– Leads to 100-fold decrease in glycine affinity
– Greatly reduced glycine sensitivity
– Increased muscle tone, increased startle reflex
