GABA Anaesthesia Flashcards
What is the most commonly used IV induction agent?
Propofol; 200mg/20ml
Death of MJ
What is a common property of all GA drugs?
Lipophilic
Study preformed on tadpoles to determine how much GA was requried before the riting reflex was inhibited (tadpole put on back and will automatically right itself)
The amount of GA drug required directly correlated with the degree of lipophilic solubility (ability to dissolve in olive oil)
Do GAs act non-specifically via the membrane?
The more lipid the GA; the increasing anaesthetic aqueous potency
GA act by perturbing membrane structure; the anaesthetic dissolves into the lipid membrane (nerve cells) and will perturb the structure. IN perturbing the structure of the neural membrane; to produce a state of unconsciousness
Describe the alcohol cut off phenomenon in terms of GA potency
As you increase chain length of alcohol (potency as anaesthetic increases and lipid solubility increases)
However, as you start to go above C9; decanol etc the relationship begins to fall away. Lipid solubility will increase by anaesthetic potency will not
Not all anaesthetics with a potency are highly lipid soluble; particularly anaesthetic enantiomers
What is an enantiomer?
Mirror image molecules - useful compound is tomidate
Identical lipid solubility and ability to perturb membrane demonstrate a different anaesthetic potency
Can anaesthetics bind to proteins?
Yes; fire fly luciferase study demonstrated anaesthetic ability to bind to a pure protein preparation
Why protein (s) do anaesthetic bind and work to?
Transmitter gated ion channel
5 transmembrane crossing subunit
For nicotinic receptors; ACh binds extracellularly, believed 2 molecules are required for opening of the channel (cation influx)
ACh; cation influx
GABA; anion (chloride) influx
Describe the most common cys-loop receptors (nicotinic ACh)
Pentamer; 4 transmembrane proteins (TM1-4). Large N terminal, cysteine cysteine loop, extracellular carboxyl tail Nicotinic ACh - cation influx 5-HT3 - cation influx GABAA - anion (inhibitory) influx Glycine - anion (inhibitory) influx
Describe the glutamate receptor family?
Tetramers; N terminal, internal carboxyl tail
NMDA
AMPA
Kainate
Described via agonist pharmacology
What is the particular importance of TM2 of the cys-loop receptor family?
Lines the ion channel conduction region of the receptor
Describe the action of propofol on GABA inhibitory reading via patch clamp
Increases it
Describe the action of etomidate on 6 major receptors:
Inhibitory; GABAA, glycine
Excitatory; AMPA/kainate, NMDA (+ glycine), alpha4beta2 nicotinic, 5-HT3
GABAA; large increase
No action on glycine, AMPA/ kainate, NMDA, nicotnic or 5-HT3
Describe the action of propofol on 6 major receptors:
Inhibitory; GABAA, glycine
Excitatory; AMPA/kainate, NMDA (+ glycine), alpha4beta2 nicotinic, 5-HT3
GABAA; large increase
Glycine; small increase
No action on AMPA/kainate or NMDA
Decreases nicotinic moderately, slightly decreases 5-HT3
Describe the action of alphaxalone (steroid) on 6 major receptors:
Inhibitory; GABAA, glycine
Excitatory; AMPA/kainate, NMDA (+ glycine), alpha4beta2 nicotinic, 5-HT3
GABAA; large increase
No action on glycine, AMPA/kainate, NMDA ot 5-HT3
Will moderately decrease nicotinic action. Important to note that betaxolone is equieffective in this role (however this is not an anaesthetic suggesting that this action on nicotinic receptors is not involved with anaesthetic action)
Describe the action of pentobarbitone on 6 major receptors:
Inhibitory; GABAA, glycine
Excitatory; AMPA/kainate, NMDA (+ glycine), alpha4beta2 nicotinic, 5-HT3
GABAA; large increase No action at glycine receptors Large decrease at nicotinic receptors Moderate decrease of AMPA/kainate Slight decrease at NMDA and 5-HT3
Does ketamine act at GABAA?
No; inhibits NMDA receptors
Does etomidate exist as an enantiomer?
Yes; R+ and S-
R+ is used as the hypnodate
Differ in anaesthetic potency; this is via ability to modulate GABA activity
R+; as conc is increased there is a dose dependent enhancement of GABA response peaking close to GABA maximum
S- can enhance GABA but it requires much higher concentrations, with a much smaller maximum effect
Describe the GABA mimetic effect of etomidate; comparing R+ and S-
At higher concentrations required to enhance GABA; they can directly open GABAA receptor channel to cause an inward current in the absence of GABA
GABAmimetic effect much higher for R+ than S-
Describe GABAAR competitive antagonists
Bicuculline, picrotoxin
Proconvulsant and anxiogenic
Where is alpha 5 predominantly expressed?
Hippocampus
Where is the delta subunit predominantly expressed?
Cerebellum
Hippocampus
Thalamus
Does etomidate show selectivity across GABAAR subtypes?
Yes; shown to be beta subunit dependent
Specifically beta 2/3 (limited impact at beta 1)
Why does etomidate show beta subunit selectivity?
Beta 1 - no action
Beta 2 - yes
Beta 2/1 chimera; N terminal from beta 2 and intracellular/ transmembrane loop from beta 1 = no
Beta 1/2 chimera; beta 1 N terminal, transmembrane/ intracellular loop from beta 2 = YES
Critical region of TM2; single amino acid (N/S - asparagine to serine) will dictate pharmacology of etomidate
What governs the beta subunit selectivity of etomidate?
A single amino acid; N (asparagine) to S (serine) inhibits etomidate action
Is there a specific IV GA transmembrane binding site?
May concentrate in the membrane close to the transmembrane site located between beta-alpha subunit interface
Describe the binding sites for etomidate and propofol at the GABAAR
Etomidate binds at the b-a interface with the phenyl ring packed against the N265 of the beta2 subunit
Propofol binding overlaps with etomidate but due to the small nature of the molecule; makes fewer connections with the receptor
What is the influence of etomidate and propofol at the inter-subunit binding cavities?
In the presence of GABA there are 5 interfacial sites; which are all open
In the presence of etomidate (alpha-beta2 cavity) it will close 2 of the cavities (unbound)
Propofol also binds at inter-subunit binding cavities; but again due to the small nature of the molecule it fails to close any interfacial sites
What did the work surrounding the beta unit sensitivity of etomidate lead to?
Creation of a beta subunit “knock-in” etomidate insensitive mouse
In vitro; Beta 2 (N265S) = reduced etomidate sensitivity Beta 3 (N265M) = reduced etomidate and propofol sensitivity
Does etomidate produce sedation in beta N265S mice?
No - look at locomotor activity in mice as a surrogate measure of sedation
Still possible at high doses to produce hypnosis
Can be argued that locomotor activity proves a limited surrogate for sedation
Slow wave sleep in beta2 mutant mice by etomidate was greatly reduced
What is the loss of righting reflex (LORR) used for as a model?
Surrogate measure for unconsciousness “anaesthesia” in humans
Are the LORR effects via etomidate affected by mutant B2 or B3 mice?
Yes; the LORR by etomidate is reduced in both the beta 2 and beta 3 mutant mice
Therefore for LORR; etomidate acts at beta 2 and beta 3 receptors
Why is LORR considered a good surrogate for anaesthesia in humans?
There is a correlation between the anaesthetic dose required to cause loss of consciousness in 50% (EC50) of humans (failure to respond to a verbal command) and the dose required to produce LORR in rats an mice
How is immobility and surgical tolerance determined in mouse models?
Tail withdrawal reflex
Are the immobilising effects of etomidate (& propofol) affected by beta mutant mice?
Yes; beta 3 N265M mouse shows a blunted immobilizing effect but NOT beta 2N265S mice
Therefore, the immobilizing effects of etomidate and propofol are mediated by beta 3
By use of the beta mutant mice models; what effects of etomidate does the beta 2 GABAAR subunit mediate?
Hypnosis in part (shared by beta 3)
Sedation
Hypothermia
By use of the beta mutant mice models; what effects of etomidate ( & propofol) does the beta 3 GABAAR subunit mediate?
Hypnosis in part (shared by etomidate)
Immobility
Respiratory depression
Describe the mode of action of etomidate
Selective, positive allosteric modulator (PAM) of GABAAR
Furthermore, highly selective for GABAAR incorporating beta 2 or 3 subunit vs those containing beta 1 subunit
This beta unit selectivity is governed by the nature of a single amino acid (asparnine for beta 2 and 3 and serine for beta 1)
What mediates synaptic and extrasynaptic GABAARs respectively?
Synaptic; phasic inhibition
Extrasynaptic; opened by low concs of GABA, slow tonic current
Do GAs act at the thalamus?
Yes; shown to be highly inhibited by propofol
Describe the innervation to the ventrobasal (VB) nucleus of the thalamus
Inhibitory GABA from nucleus reticularis (nRT) Sends excitatory (glutamergic) to the nRT and cortex (layer 4)
Cortex (layer 5 and 6) sends glutamergic signals back to the nRT and to the VB neurons
From a variety of different mice (WT and many variant mutant mice) what can be deduced about thalamocortical (VB) neurons via patch clamp recordings?
Synaptic GABAARs alpha 1, beta 2, gamma 2
Extrasynaptic GABAAR alpha 4, beta 2 and delta (BZD insensitive due to alpha 4 and delta)
Both phasic synaptic and tonic extrasynaptic receptors express beta 2 subunit (etomidate sensitive) `
What are the effects of etomidate on the synaptic GABAAR of thalamocortical neurons?
Synaptic alpha 1, beta 2, gamma 2 WT mice demonstrated a mIPSP that was prolonged by etomidate
This prolongation was completely prevented in mt Beta 2 (N265S) mice
What are the effects of etomidate on the extrasynaptic GABAARS of thalamocortical neurons?
Alpha 4, beta 2, delta WT mice demonstrated a tonic current, etomidate greatly enhanced this. Furthermore, around 86% of the effects of etomidate are mediated by this tonic, extrasynaptic current
This effect was completely blocked in the mt beta 2 (N265S) mutation
Are the sedative effects of etomidate mediated by is synaptic or extrasynaptic action?
Extrasynaptic receptors - modelled by gaboxodol whereby a delta -/- and alpha 4 -/- mice show no effects to gabxodol
Sedative effects are mediated by enhancement of tonic inhibition via GABAA extrasynaptic delta receptors
Describe gaboxadol (THIP)
Rigid analogue of the GABAAR agonist muscimol
Promotes a natural sleep pattern (increases slow wave sleep on EEG recordings)
Exhibits little rebound insomnia on withdrawal
No interaction with ethanol or BZD
Thalamic extrasynaptic alpha 4, beta 2, delta GABAAR are the gaboxadol target
Describe the behavioural effects of THIP in the delta -/- mouse
Decreased ataxia
Decreased sedation
Decreased analgesia
Do thalamic neurons respond to single action potentials or different firing rates?
Firing rates:
Tonic firing
Burst firing
Describe the differences between tonic and burst firing
Tonic firing; 25 Hz; dominates in waking
Burst firing; 200-400 Hz; predominates during periods of drowsiness and NREM sleep
Describe the action of DS2 on inhibitory GABA signals
DS2 is a selective PAM of delta- GABAARs
When looking at burst firing; DS2 enhances the spill over inhibition mediated by alpha 4-beta 2- delta GABAARs
This effect is absent in alpha 4 -/- neurons
What are the 3 forms of thalamic inhibition
Phasic
Tonic
Spill over - related to the rate at which the presynaptic nRT neurons are firing
Describe the effects of propofol/ etomidate on phasic, tonic and spill over inhibition in the thalamus
Prolongs phasic
Increases tonic
Greatly prolongs spill over inhibition
Describe the effects of THIP (GABAAR agonist) on phasic, tonic and spill over inhibition in the thalamus
No effect on phasic inhibition
Prolongs tonic inhibition (directly activates extrasynaptic receptors)
No research on spill-over but hypothesized to have no effect (this is mediated by activity dependent release of GABA engaging extra-synaptic receptors)
Describe the effects of DS2 (selective PAM of delta-GABAAR) on phasic, tonic and spill over inhibition in the thalamus
No effect on phasic inhibition
Greatly increases tonic inhibition
Prolongs spill over component
Is spill over inhibition important for the effects of etomidate and propofol?
In vivo study (mice) suggest that the spill over component is important in mediation of EEG signature of anaesthetic hypnosis
Record EEG of free moving mice, and then to see what the application of etomidate/ DS2 do to that EEG signature
NREM sleep triggers an EEG signature of anaesthetic hypnosis (NREM sleep assoc with burst firing of nRT neurons releasing bursts of GABA onto VB neurons)
Etomidate (or DS2) in the thalamus enhances alpha-beta activity only during NREM sleep.
What is the significance of alpha-beta frequency in the frontal cortex?
Increased alpha-beta frequency at the onset of behaviour correlates with the loss of consciousness assoc with propofol/etomidate
Furthermore, elevated alpha-beta activity in the thalamus precedes similar activity in the cortex
What is the impact of etomidate, DS2 and THIP on alpha-beta activity in the thalamus?
Etomidate (or DS2) in the thalamus enhances alpha-beta activity only during NREM sleep
THIP has no impact
Interestingly, this effect did not occur in delta -/- mice
This suggests that during NREM sleep state that etomidate induces, there is enhancement of GABA spill over leading to thalamocortical oscillations in the alpha - beta range
Which GABAARs have been implicated in post anaesthesia-induced impaired cognition?
Hippocampal alpha 5
37% of young adults and 41% of elderly patients exhibit postop cognitive impairment at hospital discharge, with 6% of young adults and 13% of elderly still exhibiting deficits at 3 months post surgery
Where are alpha 5 GABAARs located?
Extrasynaptically in CA1 neurons of the hippocampus
Therefore mediate a tonic conductance
Are alpha 5 GABAARs BZD sensitive?
Yes - interface of alpha and gamma
Describe the differences in tonic inhibition mediated by alpha 5 and delta subunits respectively
Delta = insensitive to BZD
Alpha 5 = enhanced by diazepam (PAM) and decreased by alpha 5- NAMs
Describe PAMs, NAMs and antagonists of the BZD binding site
PAM (diazepam, flunitrazepam) = enhancement of GABA response (anxiolytic, sedative, analgesic and anticonvulsant)
NAM (DMCM = beta-carboline) = decrease GABA function (pro-convulsant, anxiogenic)
Antagonist (flumazenil) = don’t influence GABAAR function, but prevent the action of PAMs and NAMs
All these drugs bind at the interface of the alpha-gamma interface
Alpha 5 NAMs exhibit pro-cognitive properties but are not convulsant/anxiogenic in comparison with NAMs that act on alpha 1,2 and 5
What is the effect of etomidate and diazepam of LTP?
They supress LTP in WT but not alpha 5 -/- mice
This suppression of LTP is prevented by the use of alpha 5- GABAAR NAM L655,708
What is the effect of alpha-5 NAMs on etomidate impaired LTP?
Alpha-5 NAMs can rescue etomidate impaired LTP
They bind to the alpha-5, beta, gamma 2 GABAARs at the BZD site to decrease the GABA evoked response
How can cognition be measured in rodents?
Novel Object Recognition Task (NOR)
Mice will naturally explore novelty and find it rewarding
Is there an effect on NOR task in mice with etomidate administration?
NOR performance was compromised by a sedative (8mg/kg) dose of etomidate for 3 days post anaesthetic, but recovered by 1 week
NOR performance compromised by an anaesthetic (20mg/kg) dose of etomidate for 1 week, but recovered by 2 weeks
Performance not confounded by sedation, or by reduced exploratory behaviour
Memory impairment can be prevented by treatment with alpha 5 NAM 30 mins before the training. These deficits induced by etomidate did NOT occur in alpha 5 -/- mouse
Summarise the prolonged memory impairment via GA
NOR performance was compromised by a sedative (8mg/kg) dose of etomidate for 3 days post anaesthetic, recovery by 1 week
Hippocampal LTP still reduced 1 day after 8mg/kg etomidate, recovery by 1 week. Hippocampal LTP suppression does NOT occur in alpha 5 -/- mouse
CA1 tonic current mediated by extrasynaptic alpha 5 GABAARs was greatly increased at 1-3 days (post etomidate) with no effect on amplitude, frequency or time course of mIPSCs mediated by synaptic GABAARs
This increased tonic current was mediated by alpha 5 GABAARs as it was blocked by the NAM L655-708
Furthermore this effect of etomidate does not occur in alpha 5 -/- mice
These prolonged effects of etomidate on tonic current are replicated by an anaesthetizing dose of inhalational anaesthetic isoflurane, which is undetectable in the brain 24 hours post treatment
How do etomidate and isoflurane produce such a persistent effect on the tonic current, when the anaesthetic is eliminated and therefore the GABAAR modulatory effects are long gone?
In the hippocampus, etomidate and isoflurane will selectively increase the cell surface expression of either alpha 5 or beta 3 subunits, with no effect on the synaptic alpha 1 or e.synaptic delta subunits. This was determined 24hrs post anaesthetic
The anaesthetic increases cell surface expression of these extrasynaptic receptors - increased tonic current
IN CONCLUSION: brief exposure to the GABAAR GA selectively increases cell surface expression of hippocampal alpha 5 GABAARs resulting in an increased tonic current and cognitive effects
Is there an involvement with inflammation (IL-1beta) with the memory impairment triggered by GA?
Inflammation triggered by surgical trauma appears to contribute to POCD in rodents as determined in NOR test and fear assoc memories
Inflammation results from autoimmune diseases, infection, TBI, stroke can lead to memory impairment
Inflammation also contributes to chronic neurodegenerative diseases such as AD, PD, MS and AIDS related dementia
Systemic inflammation increases production of multiple cytokines in the brain including IL-1 beta
In patients with sepsis induced encephalopathy, there are increased plasma levels of IL-1B correlated with cognitive deficits
In animals undergoing surgery, elevated levels of hippocampal IL-1B correlated with memory deficits
Describe the contextual fear memory test
Hippocampal associated test
Exposed a control mouse to a mild electric shock. 24hrs later they put the mouse back into the cage. If the mouse remembered this unpleasant experience, this mouse demonstrated a freezing behaviour, however if there is no recollection on 2nd exposure the mice will demonstrate normal behaviour
What study was performed to show that alpha-5 GABAARs regulate inflammation induced impairment of contextual fear memory?
Contextual fear memory test
Mice treated with IL-1B to mimic acute systemic inflammation
Inflammation induced mice did not show freezing behaviour, impaired fear memory
If the IL-1B mouse was given alpha 5 NAM 30 mins before training, the mouse showed freezing behaviour
Furthermore, alpha 5 -/- IL-1B mice exhibit normal recall of the prior event
Describe the impact of IL-1B on hippocampal LTP
WT treated with LPS (increases IL-1B) decreased hippocampal LTP; an effect prevented by alpha 5 NAM, and was not evident in alpha 5 -/- mice
IL-1B increases the magnitude of the hippocampal CA1 tonic current of WT but NOT alpha 5 -/- mice.
To note, IL-1B has little effect on mIPSCs (synaptic GABAARs)
In hippocampus, IL-1B increases the surface expression of alpha 5 GABAAR subunits
In the hippocampus, IL-1B and etomidate act synergistically to greatly increase the tonic current
Describe the BZD binding site on GABAARs
Between alpha- gamma subunit interface
For high affinity binding of diazepam, the receptor must contain a gamma subunit (usually gamma-2)
GABAARs incorporating gamma 2, alpha 1,2,3,5 are BZD sensitive
GABAARs incorporating gamma 2 with alpha 4 or 6 are BZD insensitive
Can BZDs be used as analgesics?
Intrathecal diazepam suppresses the sensitized response to heat and mechanical stimulation evident in rodent models of both inflammatory and neuropathic pain
How can analgesia be measured mouse models (inflammatory pain (zymosan A injection into left hind paw) and neuropathic pain (tie off sciatic nerve))?
Heat hyperalgesia - paw withdrawal latency upon exposure to a defined radiant heat
Mechanical sensitization - paw withdrawal with Von-Frey filaments
Cold allodynia - time spent lifting, shaking or licking the paw after a drop of acetone on the injured paw
Using these mouse models of inflammatory and neuropathic pain, what were the effects of intrathecal diazepam?
Inflammatory pain and heat hyperalgesia; diazepam is analgesia; primarily mediated via alpha 2/3 GABAARs
Neuropathic pain and heat hyperalgesia; diazepam promotes analgesia primarily via alpha2/3
Are alpha 2 GABAARs a novel analgesic target?
Alpha 2 GABAARs are densely expressed in the mouse lamina 2/3 dorsal horn neurons and primary afferent DRG
Intrathecal diazepam supresses the sensitized response to heat and mechanical stimulus in inflammatory and neuropathic mouse models
These effects of diazepam are maintained in alpha 1 H101R but NOT alpha 2 H101R mice
In rats, a compound that enhances alpha 2,3 and 5 (but little on alpha 1) supresses the sensitization (mechanical and heat) evident in both inflammatory and neuropathic models. In contrast to morphine, this specific GABA receptor did not lose efficacy (develop tolerance) over a 9 day time period
Can alpha 5 NAMs be used as cognitive enhancers?
BZDs (PAM) impair cognition, particularly when combined with alcohol
However, NAMs of the alpha 5, beta, gamma 2 subtype that bind to the BZ site between the alpha 5-gamma 2 subunit interface exhibit precognitive properties in mouse models of:
HD
AD
DS
Impaired cognition assoc with neuroinflammation and post anaesthesia
Describe the specific of GABAAR subunits and their behavioural effects
Alpha 1 = sedative
Alpha 2 = anxiolytic, analgesic
Alpha 5 = impaired cognition
