447 final new stuff

mansvelder & McGehee paper

long term potentiation of excitatory inputs to brain reward areas by nicotine

self administration of nicotine

• 6 OH dopamine lesions of dopaminergic terminals in the Nuc Ac extinguishes nicotine self administration

• placed a catheter in jugular vein of the rat and had syringe filled with nicotine

• when they pressed this active lever this would self administer nicotine in its jugular vein

• when they gave the rats nic with no toxin they were self administrating nicotine

• gave it to them with toxin they didn’t self administer nicotine

• this indicated the dopaminergic pathway is important for mediating the reinforcement of nic as a drug

mesolimbic pathway

DA neurons in the VTA project to the nucleus accumbens, prefrontsl cortex, olfactory tubercle, amygdala, and septal region

dopamine: prediction and assessment of reward

• monkeys were given squirt of juice at random times

• if the monkey receives juice unexpectedly the DA neurons fire a lot

• prepared a conditional stimulus with that reward, the DA neurons did not have firing over that reward but did for the anticipatory stimulus

• reward predicted and no reward occurs, DA neurons fired after conditioned stimulus, when it doesn’t receive reward DA neurons decrease in firing

• activity of DA neurons were dependent on the context of the reward

nic injection increases dopamine for hours

• did micodialysis recordings from rats, sampled the extracellular solution and measured the concentration of dopamine in the extracellular solution in the brain

• can see after injection of nic and once nic is injected there is a spike like increase of dopamine levels in nucleus accumebns

• can see it goes down but remains elevated for 3 hours

long term potentiation

• learning and memory is believed to be encoded by the strengthening of synapses

• LTP was discovered in 1996 by Terje Lomo in the hippocampus of the rabbit

• long term enhancement of synaptic transmission occurs if there is an increase in synchronous activity of pre and postsynaptic neurons

• time scale of long term is greater than 1 hour

• requires presynaptic release of glutamate

• requires activation of postsynaptic NMDA receptors

• requires a rise of intracellular Ca thru NMDA receptors

• intracellular Ca activates CaMKII

• CaMKII phosphorylates AMPA receptors

• AMPA receptors translocates to the cell surface membrane

Manvelder and McGehee 2000: Goals of study

• concentrations of nic that are reached in the blood of smokers depolarize DA VTA neurons by activating nAChRs on the somata of these neurons. at these concentrations, nAChRs also undergo desensitization within secconds, yet nic injections in rats enhance DA release in the NAcc for more than one hour

• additional mechanisms must contribute to the prolonged effects of nicotine

• they examined a potential mechanism of nicotine addiction due to altered neuronal activity of dopaminergic neurons in the VTA

• they examined how nicotine exposure can modulate the plasticity of excitatory synaptic transmission of dopaminergic neurons in the VTA

they did what type of recording on what animal

they did a whole cell patch clamp electrophysiology on the brain of rats

nicotine enhances evoked EPSC to DA VTA neurons

• one of the difficulties in recording in VTA have 2 neurons, DA and GABAergic

• DA neurons have an IH current- this is a hyperpolarization activated cyclonucleotide gated channel

• hold this at -60mV and they do step hyperpolarization all the way to -120mV build up of inward current

• saw that nicotine is acting presynaptically

nicotine enhances spontaneous EPSC freq of DA neurons

• appllied nic there is more frequent glutamate release

• there is cluster of sEPSC

• washed off the nic and it went backto baseline

• put nic receptor inhibitor MEC for a4b2 receptors

• put MLA which is specific for alpha 7 nic receptors

• MEC inhibits a4b2 on the soma, but on the presynaptic nicotinic receptors that enhance glu release these are insensitive to MEC

• wanted to know whether this increase in freq was due to increase in AP freq and therefore AP dependent

• can test this pharmacologically can block AP by blocking Na channels

• TTX is one Na channel blocker, when they applied nic they still saw an enhance in the frequency of glu release this is AP independent

a7 nAChRs enhance spontaneous EPSC frequency

• can see it is a7 mediated, MLA inhibitor abolishes this enhancement of EPSC

• in the presence of MLA it completely inhibits it

• presynaptic is a7 not a4b2

• MEC is more sensitive to a4b2 but when you get to higher concentrations it can start inhibiting a7 it is not so selective like MLA

Induction of LTP by presynaptic stimulation with nicotine

• didnt do whole cell recordings did perforated patch recordings

• patch electrode is filled with solution that mimicks the intracellular but it is only ions

• patch clamp use amphotericin-b toxin this goes into the membrane and makes very microscopic pores in the membrane and this allows the researcher to have electrical connectivity in the cell but it won’t dilute intracellular contents of the cell

• nicotine can induce lTP when paired with post synaptic depolarization

presynaptic depolarization and nicotine does not induce LTP

• looked at presynaptic stimulation and applied nicotine but don’t apply post synaptic depolarization there is no induction of LTP

• during presynaptic stimulation they did not clamp postsynaptically

a7 nAChRs and NMDA receptors contribute to LTP

• nicotine with postsynaptic depolarization this told them this was through a7

• not only is it through a it is also with NMDA to get LTP

summary

• LTP in dopaminergic neurons was facilitated when nic was coapplied during postsynaptic depolarization

• LTP is dependent on a7 mediated enhancement of glu release

• LTP is dependent on activation of postsynaptic NMDA receptors

• presynaptic a7 nAChRs enhanced glutamate release onto dopaminergic neurons

Labarca et all 1995

channel gating governed symmetrically by conserved leucine residues in the M2 domain of nic receptors

what is a gate?

looks like a door that closes, a barrier for ion flow

What is gating

the mechanism for which ion channels open and close

nAChR structure

• nicotinic receptors are part of cys loop and pentameric receptor family

• M2 is pore lining region

• they investigated a leucine in M2 region and that is involved in gating of the channel

structure of nAChR known in 1993 at 9A resolution

• kink like structure on the M2, this is pore lining domain

• at the 9’ position this is well conserved not only in nAChR but also cys loop family receptors

• all the ones in the prime position are all hydrophobic and they all line up

• right beside the hydrophobic aa that line up there are hydrophilic aa that line up

oily knee hypothesis

• leu 9’ is the M2 “kink” in unwins structure of nAChr

• oil is hydrophobic and knee has a kink

• have 5 subunits where kink is pointing towards center of pore and this provided a steric hindrance and it is very constrained at this position

• the leucine side chain at the kink is pointed towards the center of that pore and therefore ions cannot flow thru

• when the gate opens it twists and turns so leucine moves away from the pore and the hydrophilic aa line up at the pore

labarca et al 1995: goal of study

explore the functional role of the residue, leu 9’ which is located midway up the M2 helix in the muscle nicotinic receptor

dose response relations of Leu9’Ser mutations

• expressed mRNA in xenopus oocytes and did 2 electrode voltage clamp recordings

• by the time of 4 mutations a mutant only needs 1 Ach bound to open up receptor where wild type needs 2 Ach to open up receptor

• it was thought that alpha subunit was key subunit for gating but they found if you mutate any subunit this decreases the EC50

single channel recordings of L9’S

• alpha 2 beta gamma delta had three fold longer open times than wildtype nAChRs and had much longer bursts (lasting hundreds of ms) than wildtype

summary

• leu 9’ ser results in an increased apparent sensitivity to ACh

• 10 fold decrease in EC50 with each subunit mutated

• Leu 9’ ser results in longer single channel open times and burst duration

• leu makes the channel more difficult to open

Kearney et al 1996 neuron: futher analysis of 9’ M2

• amino acids at 9’ with greater hydrophobicity are more difficult to open

• as you get to greater hydrophobic amino acids, you get higher EC50, it is more difficult to gate open the receptor

Akabas paper

• proped the amino acids of mouse muscle nAChR in M2

• did a site directed mutagenesis, electric physiology and cross linking

• mutated Leu 251, which would be the 9’ position of the alpha subunit

• mutated it to cysteine because they applied MTSEA, if the side chain if leucine-cysteine is accesible this MTSEA will form a disulfide bond and it is large enough that it will block the pore

• they applied crosslinking chemical MTSEA with Ach to see its effects while channel is open

• when MTSEA is applied in activated state, MTSEA has access to 9’ position and crosslinked and inhibited current because it blocked ion flow

effect of MTS on M2 residues in closed and open states

• there is a structural movement of M2

• L251, which was not accesible from the pore at the resting state is accesible or faces the pore when activated by ACh

• since every second residue faces the pore (accessible by MTSEA) then M2 must be a B strand structure

• they proposed that Leu 9’' did not face the pore in the closed state it faced away from the pore, once it is activated there is change in conformation for it to bind to the side chain so likely it changes the center of the pore

• they proposed that side chains faced the pore every second amino acid, this means that M2 must be a beta strand structure (WRONG)

effect of QX-222 on M2 residues in closed and open states

• since every fourth residue faces the pore (is accessible by QX 222) then M2 must be an alpha helix structure

• mutated all the different subunits and started mutating all amino acids along M2, mutations to 2’, 6’ 10’ affected the affinity of binding to QX-222

• only these amino acids face the pore that affect binding

• because it is every 4 amino acids, it is not a beta strand structure it is an alpha helix structure

• this leucine 9’ position and they said that the side chain does not face the pore it faces away- agrees with akabas

• had different secondary structure for M2, akabas said it was beta strand

Cryo EM structure of Torpedo nAChR

• side chain of leu is not pointing towards center of pore, it is pointing towards neighboring side chains of alanine, there is hydrophobic-hydrophobic interaction between Leu and Ala

• Leu 9’ (L251) MAKE SIDE TO SIDE HYDROPHOBIC INTERACTIONs with neighboring alanine produces a tight hydrophobbic girdle

• Leu 9’ (L251) and val 13’ (V255) form two hydrophobic rings which would be the effective gate of the channel

• they would form an energetic barrier to ion permeation

• cys loop comes close to the membrane and interacts with the receptor at M2-M3 loop

bacterial pentameric ligand gated ion channels

• very similar to nAChR structure

• no double cys in that C loop

• don’t have a cys loop

• have a loop that comes close to membrane but no disulfide bond

• these bacterial channels are not cys loop receptors but are members of pentameric ligand gated ion channel family

ELIC and GLIC

• ELIC is closed conformation

• GLIC is open, sensitive to protons, low pH will open up the channel

• GLIC doesn’t have leucine in 9’ bit it has isoleucine, the conformation is somewhat changed it no longer has bow like shape like ELIC it is straightened out

• shows that this gating involves subtle conformational change, go from bow shaped to kinked M2

• the distance between M2 is larger when it is straightened out vs when it is bow shaped and bent and closed

cryo-EM structure of nicotinic ACh receptor

• M2 appear to straighten while moving out in open position

• when Ach is bound, the alpha subunit is straightened out and is parallel to straight dashed line and the opening is wider as it goes from bow shaped to straight

• while nAChR gate open there is a conformational change that it straightens out and it goes further away from each other and allows ions to flow thru, no ligand bound it is bent bow shaped and kinked and the distance between the subunit is less

lummis et al 2005

cis trans isomerization at a proline opens the pore of a neurotransmitter gated ion channel

serotinergic related disorders

• serotonin (5-HT) is a major neurotransmitter in the CNS and found in the raphe nucleus

• the serotinergic system is implicated in a variety of emotional states, mood and mental arousal

• a polymorphism in the 5-HT3A gene is associated with schizophrenia

• 5-HT system is targeted for treating depression and anxiety

• dysfunction of 5-HT3 receptors has been implicated in IBS

high frequency HTR3B variant associated with major depression dramatically augments the signaling of the human 5-HT3AB receptor

• Y129S polymorphism in the 5-HT3B receptor is inversely correlated to major depression

• one of the polymorphism is amino acid tyrosine 129, this individual is more susceptible to major depression

• have a serine they are less afflicted for major depression

• serine desenstized 10 times slower than tyrosine

• did cell attached patch clamp, there is a gigaohm seal, the binding site is in the patch pipette, they put 300 mM serotonin into the patch pipette so it will activate the receptor and looked at the channel activity

• the serine mutation is open for longer time, it deactivated a lot slower, there is stronger serotonergic signaling which leads to a decrease in susceptibility from major depression

Lummis et al 2005: Goal of study

• the neurotransmitter binding site in the cys loop superfamily of ion channels is located about 60A from the channel pore, presenting a conundrum as to the molecular events that link binding and gating

• evidence implicates the M2-M3 loop and in low resolution structural studies this region interacts with loop 2 and 7 of the extracellular domain

• in the cation selective nACh and 5-HT3 receptors the apex of the M2-M3 loop contains a conserved proline that is ideally placed to provide a hinge for movement of the channel lining M2 helix

• the study sought to examine the functional role of proline in the gating of the 5-HT3 receptor

amino acids prefer the trans conformation

steric hindrance does not favour the cis conformation in peptide bonds

prolines

• the cis and trans forms can interconvert

• proline has cyclic pentameric structure, has more propensity to be in cis conformation

• proline is 100 times more likely to be in cis conformation than trans conformation

• proline is amino acid where they bend because they can transition from trans to cis

• during gating it switches from trans to cis

• did site directed mutagenesis from proline to other aa, the protein folded and was able to bind to agonist but it was not functional

unnatural amino acid incorporation into ion channels

• took the mRNA from 5-HT3A receptor and expressed both 5-HT3A and 5-HT3B

• chemically attach unnatural amino acid to express it and replace proline

• inject tRNA with unnatural amino acid and inject it in xenopus oocyte and did voltage clamp recording

unnatural amino acids and their cis trans preferences

• pro- 5

• pip-12

• aze-18

• Tbp-55

• Dmp-71

• high propensity for cis the EC50 lowers

relationship of cis preferences to receptor activation

• did a binding assay with radioactively labelled antagonist and found no difference in mutated and wild type proline

• found when they did a linear regression, line fits very nicely through the points and the slope is really close to 1

• they concluded that the energy difference between the transition from trans to cis during gating is fully imparted (fully put into the gating process) to the energy needed to gate the receptor EC50

• proline 8* played a critical role in gating based on what is shown in this relationship

• key is this transition from trans to cis

current traces of 5-HT3 receptors with unnaturals

• 5-HT3 receptors with Dmp at 8* activate with 5-HT but do not fully deactivate

• Dmp is the one that transitions to cis 71% of the time

• there is irreversible activation of the channel

• they put a channel blocker and they are able to inhibit almost all channel activity

2 conformations found of M2-M3 peptide

• the major form are 5X more prevalent than minor form

• major form: trans conformation of Pro 8*

• Minor form: cis conformation of Pro 8*

proposed gating mechanism of 5-HT3 receptors

• in closed conformation proline is in trans conformation and it is in apex of M2-M3 and when it is in trans conformation the channel is closed

• loop 2 and loop 7 on either side of proline

• they proposed in the binding pocket there is the key residue TrpB it is several residues down loop 7 and causes a conformational change in loop7

• there is the C loop this moves and closes on the agonist

• when this C loop moves in it also effects the structure of these loops and causes it to move

• the loops can no longer constrain proline in trans conformation and then it transitions to cis conformation and is at apex on M2-M3

• acts as a gating hinge, bends the M2 and allows for ion flow

summary

• loops 2 and 7 acts as calipers to lock Pro8* in the trans or closed conformation

• upon ligand binding loop C closes in and likely dislodges the loop 7 caliper so that Pro 8* is now free to isomerize to its cis conformation

• the transition of pro 8* to its cis transformation causes a bending and rigid movement of the M2 alpha helix to open the pore

proposed gating mechanism of nicotinic receptors

• examined not only pro 8* but also residues on loop 2, B10 strand and residue on top of M2 and their interactions

• interaction between innerphase and transmembrane region and this involves the following:

  • loop 2 has glu and interacts with arginine positive region on B10 and forms a salt bridge

  • look at interaction of proline and valine and serine since it is close proximity to loop 2

loss of function as salt bridge disrupted in loop 2 & B10

• did a mutation to break the salt bridge

• mutated arginine and this shows that opening is briefer

• mutated to lysine and has shorter duration

• they did a double mutation to maintain the salt bridge, made glu M2 to arg and arg b10 to glu and they got a rescued response and open duration this is very similar to wild type

• salt bridge between b10 strand and loop 2 is important for gating

Val 46 in loop 2 interacts with Pro8* and Ser269

• mutated proline to glycine and got a shorter opening

• proline needs to interact with loop 2

• mutate ser to leu they change the open duration

• same with mutation of valine they change the open duration

• you need precise positioning of these loops and b10 strand in a certain position to have normal gating

summary

• there is a critical interaction between Pro8* on M2-M3 and the hydrophobic residue Val46 on loop 2

• the salt bridge between glu45 on loop 2 and Arg 209 on B10 strand is vital for normal function

• Val46 also makes important interactions with the top of the M2 through Ser269

Protomer structure of AChBP

• consists of N terminal alpha helix, two short 310 helices and 10 b strands forming beta sandwhich

• N terminal at top and c terminal at bottom

Kash et al 2004

coupling of agonist binding to channel gating in the GABAA receptor

subunit stoichometry of GABAA receptors

• consists of 2 alphas, 2 betas and 1 gamma subunit

• there are a total of 19 GABA r subunits

• GABA binds at the alpha and beta interfaces to open the channel which fluxes inward Cl-

benzodiazepines

• cannot open up the channel but binds somewhere else to modulate the channel and when gaba binds the current is increased

• another binding site for benzos which targets gabaa receptors, they are not agonists but increase gabaa current and increase it by being a positive allosteric modulator

GABAA receptor related channelopathies

• mutations to GABAA subunits including gamma 2, alpha 1, beta 3 and delta result in a variety of epilepsies

• single nucleotide polymorphisms in the GABAA B2 subunit is associated with schizophrenia

• two single nucleotide polymorphisms in the GABAA gamma 3 subunit is associated with autism

• the gene for GABAA alpha 6 subunit is identified as an inheritable locus for alcohol dependence

• bezos target gamma containing gabaa receptors in the treatment of acute anxiety

first genetic evidence of gabaa receptor dysfunction in epilepsy: a mutation in the gabba 2 subunit gene

• K289M mutation results in generalized epilepsy with febrile seizures

• lots of members of the family had generalized epilepsy with febrile seizures

• found a common mutation of GABAA gamma 2 subunit gene

• instead of lysine it is mutated to methioinine, goes from + charge to one that is hydrophobic

• it is in the M2-M3 loop, the lysine is conserved throughout all these subunits

effect of K289M mutation of GABAA gamma 2 subunit

• they did electrode voltage clamp recordings of the GABAA subunits expressed in ooxytes

• when they applied 1 mm GABA they get this inward current

• the mutant they mutated lysine to methionine that is found in patients with epilepsy and can see the GABA current is quite smaller

• a decrease in this inhibitory current would cause excessive excitation in neurons and lead to epilepsy

Kash et all 2004 goal of the study

• many inherited mutations in the transmembrane 2-3 linker region of LGICs alter gating and are associated with human diseases

• the clustering of gating mutations in the 2-3L region has led to the suggestion that this loop is essential for communicating conformational changes that result from interaction of NT with the ligand binding site to the transmembrane domain and its integral ion channel

• homologous loops in the GABAA-R would be positioned close to the interface between the extracellular domain and the membrane, enabling loops 2 and 7 to interact with the 2-3L region

• tested the hypothesis that electrostatic interactions between charged residues in loops 2 and 7 and those in the 2-3L region contribute to GABAA-R gating

sequences of GABAA loops 2,7 and M2-M3 loop

• M2-M3 linker has + charged aa

• loop 2 and loop 7 have negative charged aa

• this provides opportunity for salt bridges to form

charge reversal mutation

• changed it from lysine + to aspartic acid -

• they thought this would disrupt the neg charged interactions

• see decrease in functionality has less sensitivity to GABA

• they mutated lys to asp and on the cys loop did a charge reversal mutation and this seems to have rescued the sensitivity to GABA but see maximal response closer to 3mM

charge exchange support loops 2,7 and M2-M3 interactions

• proposed that these aa make electrostatic interactions

• used mutant cycle analysis

• didnt measure EC50 but measured the binding affinities to protein and determined the coupling energy

• this told them that likely in GABAA receptor these 2 asp must be between 5A distance in M2-M3 loop and that is what enables it to have strong coupling energy

crosslinking supports loops 2,7 and M2-M3 interactions

• mutated asp57 to cysteine and corresponding lysine to a cysteine and if they apply an oxidizing agent those cysteine will form a disulfide bond

• oxidizing agent was Cu-phenanthroline to initiate disulfide formation

• reducing agent is DTT

• applied gaba with oxidizing agent you can see there is an inhibition of current

• this told them that loop 2 aps 57 was close enough to lysine M2-M3 in resting state such that it is mutated to cysteine it will cross link and cause an inhibition of the current

• this suggested in the resting state loop 2 is within 5A of M2-M3 lysine so there is strong coupling energy between them

• when you gate the channel this loop 7 moves closer to M2-M3 and this is how the conformation change is transmitted

proposed gating mechanism of 5-HT3 receptors

• have gate M2 leu 9’

• on apex of M2-M3 linker have proline which is gating hinge and can bend M2

• have interaction between loop 2 and loop 7 and they act like calipers on either side of proline and lock it into trans conformation

• when agonist binds the C loop closes in and Trp on B loop several residues down and it binds to agonist and it likely causes a movement in loop 7 somewhat closer to M2-M3

• change in conformation in loop 2 and loop 7 which allows it to transition from trans to cis

• this M2-M3 bends along with proline and moves leucine out of the way for ion flow

Summary

• in GABAA-R there are critical electrostatic interactions between acidic residues on loops 2,7 and lys 279 on the M2-M3 loop that are required for normal gating of the receptor

• similar negatively and positively charged residues are localized on the muscle nicotinic receptor indicating similar mechanisms of gating

• however, no basic residues on the M2-M3 loop of 5-HT3 and some neuronal nACh receptors suggesting different mechanisms of gating

• GABAa, glycine, 5-HT3 and nAChRs all contain Pro 8* on the apex of M2-M3 loop and all likely involve trans-cis isomerization during gating

Tapia et al 2007

Ca permeability of the (14)3(b2)2 stoichometry greatly exceeds that of (a4)2(b2)3 human acetylcholine receptors

goals of the study

• examined how different stoichometries and subunits of nicotinic receptors influenced calcium permeability

• examined whether a glutamic acid on the exterior of M2 was responsible for calcium permeability

M2 region of a4 and B2 nicotinic subunits

• a4 has a glutamate near the end of M2 and for b2 it has a lysine instead

• 3 a4 should be more calcium permeable than 2 a4

change in nicotinic reversal potential by 10X increase calcium concentration

• B-6-a+a4 displays the largest change in reversal potential followed by a4B2 wildtype

• B-6-a+B2 showed the smallest change in reversal potential

• more Ca permeable the effect of shifting will be greater if there is higher permeability to Ca

• alpha 7 is known as most Ca permeable and has higher Ca permeability than a4b2

• muscle nicotinic receptor has low Ca permeability

• greatest Ca permeability in alpha 7, it is greater than a4b2 but couldn’t detect significant difference there is a trend

• (a4)2(b2)2 its calcium permeability is greater than WT

charged aa’s in M2 affect Ca permeability

• nicotinic currents in normal extracellular solution was compared to that in extracellular solution where the cations Na and K other than Ca were replaced with dextrose

• neg charged aa increase Ca permeability

change in Ca permeabilities with various nicotinic subunits

• alpha 7 is known to be the most calcium permeable of all nicotinic receptors

• mutated lysine to glutamate has higher calcium permeability

• mutated glutamate to lysine there is no calcium current

• B6a+a5 is the most calcium permeable out of all the nicotinic receptors

dose response relations of WT and mutant 14B2 nAChRs

• high sensitivity is (a4)2(b2)3

• low sensitivity is (a4)3(b2)2 would be activated by high concentration of Ach

effects of mutations to M2 aa’s on Ca2+ permeability

• can see glu on inner side of the pore, mutated it and completely obliterated Ca permeability

• mutated valine to threonine this seems to increase Ca permeability

• Leu 254 and 255 mutate than and it obliterates Ca permeability

Ca permeability in muscle and neuronal nAChRs

• voltage clamp recordings of the current and simultaneously imaged Ca flux in the cells using a calcium sensitive dye

• can look at Ca influx using fluorescent dyes

Genetically encoded Ca2+ sensor: GCaMP and GECO

• some scientists have taken green fluorescent protein and fused it to a calcium binding protein like calmodulin

• linked it with another segment of protein called M13, this protein is constucted such that if there is an increase in intracellular Ca it will bind to calmodulin and there will be a conformational change that will effect the fluorescence of GCaMP

• you can have this driven by specific promotors and they imaged Ca in vivo

Cryo EM structure of Torpedo nAChR

• lots of negatively charged residues in the permeation pathway of the nicotinic receptor

• ion permeation pathway will likely go through this region

• cytoplasmic region is also lined by negatively charged residues

Summary

• there are many key residues in the M2 that control ca permeability not just the external glutamic acids

• there may also be key residues in the side portals lined by the amphipathic helix just before M4 that may control channel conductance and Ca permability in addition to the extracellular vestibule

Klaassen et all 2006

seizures and enhanced cortical GABAergic inhibition in 2 mouse models of human autosomal dominant noctural frontal lobe epilepsy

epilepsy

• 1% of population have epilepsy

• a chronic condition of the brain characterized by an enduring propensity to generate epileptic seizures, and by the neurobiological, cognitive, psychological, and social consequences of this condition

seizure

• sudden abnormal excessive or synchronous neuronal activity in the brain that transiently disrupts brain function and behaviour

• one in every ten people will have at least one seizure during their lifetime

epilepsy treatments

• carbamazepine- stabilize inactivated state of Na channels (most commonly used)

• phenytoin- stabilize inactivated state of Na channels (Most commonly used)

• valproic acid- blocks Na channels

• tiagabine- GABA reuptake inhibitor

autosomal dominant nocturnal frontal lobe epilepsy

• ADNFLE is the first idiopathic epilepsy whose genetic defect has been identified

• seizures occur during non REM stage of sleep

• nocturnal motor seizures

• it resembles as sleep terrors, nightmares or sleep walking

• EGG abnormalities may occur

• seizures originate in the frontal lobe

• autosomal dominant, only require one allele of the mutation for the individual to have epilepsy

ADNFLE

• first mutation discovered from australian family

• S252F mutation to alpha 4 nic receptor gene

• mutation to B2 nic receptor, mutations to valine

• most abundant nic receptor is a4b2 they also found a mutation in a2 subunit, this is isoleucine to asparagine mutation

ADNFLE mutation

• first mutation found is the serine 252 phenylalanine mutation

• leucine 18’ insertion mutation

• covered serine 252 phenylalanine mutation or 263 insertion mutation of leucine

nicotine patch inhibits ADNFLE seizures

• lady smoked cigarettes on medication to control these seizures

• when she was treated with nic patch the amount of seizures dramatically reduced

• nic patch stays in bloodstream for longer

• at low concentrations it desensitizes the nic receptor and quiets the activity of the neurons

Klaassen goals of the study

• examined whether 2 ADNFLE mutations in CHRNA4 S252F and L234 L18’ in humans can recapitulate the seizure phenotype when introduced into the mouse

ADNFLE knock in mouse strategy

• gene of alpha 4 nic receptor subunits it has 6 exons and 2 mutations were in second transmembrane domain exon 5

• made mutations in exon 5, have negative selection factor and positive selection factor

• through recombination they introduced the mutation back into the gene

ADNFLE mice have abnormal cortical EEG

• S252 L264 (L18’) het and hom mice exhibited higher amplitude EEG

• S252F L264 (L18’) het and hom mice exhibited increased EEG power in the delta and theta frequency ranges

• S252F het mice exhibited seizures 22% of the time

• L264 (L18’) het mice exhibited seizures 5% of the time

Nic enhances sIPSCs from ADNFLE cortex

• did whole cell patch clamp recordings from the frontal cortex

• mutations to nicotinic receptors enhanced nicotinic mediated GABAergic neurotransmission

• recording from pyramidal neurons which are glutamatergic, in the cortex have GABAergic interneurons and they synapse to pyramidal neurons to produce IPSP’s, have mutated a4 nicotinic receptors on those neurons, nicotine would act on GABAergic neurons and this would increase NT release

Nicotine enhances sIPSCs from ADNFLE cortex

• TTX and Cd decreased sIPSC frequency (27%) and amplitude (34%) and nicotine still enhanced mIPSCs in the mutants and not the WT

• the effect of nicotine is action potential independent and it mediating its effects at the terminal

• even when you don’t have AP, nic is still mediating its effects

Picrotoxin inhibits ADNFLE seizures

• use GABAa receptor inhibitor should normalize the EEG and inhibit the seizures

• injecting picrotoxin helped EEG become normal

ADNFLE mice exhibit enhanced nicotine induced seizures

• looked at mice’s sensitivity to nic induced seizures

• nic depolarizes neurons, get high enough dose can get seizures

• even further there is greater enhancement increased of sensitivity in the homozygous mice

working model of ADNFLE seizures

• in case of mutation the nicotinic receptors their activity would be greater than WT, having greater activity this would enhance GABAergic NT

• have this gain of function nic receptors which causes an excess of GABA to be released, these GABAergic neurons synapse on a number of pyramidal neurons

• normally in the cortex have asycnhronous firing of these pyramidal neurons

• once this GABAergic neurons receives cholinergic input to activate this gain of function a4 nic receptors, it will increase firing of GABAergic neurons and result in excessive GABA release, and GABAergic neurons will silence the neurons, once it stops firing the pyramidal neurons will be released of that inhibition and fire all at the same time

Teper et al nicotine induced dystonic arousal complex in a mouse line harboring a human autosomal dominant nocturnal frontal lobe epilepsy mutation- results

• S6’F ADNFLE knock in mutation in mice

• no spontaneous seizures

• no abnormal EEG activity

• enhanced sensitivity to nicotine induced seizures

• the background strain of the mice is different

• the genetic background of the individual can contribute to propensity of the individual to have seizures

Summary

• Chrna4 (S252F, S6’F) and Chrna4 (L264, L18’)

• ADNFLE mice had enhanced nicotine mediated GABAergic neurotransmission

• mechanism of ADNFLE seizures may be due to enhanced synchrony of neuronal activity as neurons recover from strong GABAergic neurotransmission

Kravitz et al

regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry

Basal ganglia

• a set of neurons in different nuclei in the brain that influences voluntary movement

• includes the caudate, putamen, globus pallidus, subthalamic nucleus and substantia nigra

• any dysfunction of neurons in this circuitry will result in a motor system that cannot switch smoothly between initiation, maintenance and termination of movement

• movement disorders involving the basal ganglia include parkinson’s disease and huntington’s disease

connections with basal ganglia

• caudate and putamen is known as dorsal striatum

• pyramidal neurons (glutamatergic) of the cerebral cortex project to medial spiny neurons of the dorsal striatum

• medium spiny neurons (GABAergic) project to globus pallidus and substantia nigra pars reticulata

• ventral midbrain there are 2 nuclei that are rich in dopaminergic neuron

• caudate and putamen contain a variety of neurons, portion of the neurons are the medium spiny neurons and they receive these dopaminergic innervations

• medium spiny neurons in the caudate putamen project to reticulata and contain GABAergic neurons

• cholinergic interneurons form a minority of neurons

Basal ganglia projections of direct pathway

• descending inputs from cortex to striatum activates MSN which disinhibit the thalamic neurons through the globus pallidus internal

• MSN and globus pallidal neurons are GABAergic

• cortical neurons are glutamatergic

direct pathway

• caudate putamen, one arm projects to internal segment of globus pallidus, projection to the thalamus and thalamus consists of glutamatergic neurons

• the direct pathway stimulates wanted movement, have globus pallidus internal segment it projects and inhibits to the thalamic nucleus, activate direct pathway by activating MSN, get disinhibition this results in thalamic neurons exciting the frontal cortex

• stimulation of MSN of direct pathway facilitates wanted movement

• have projection directly to substantia nigra pars reticulata which has GABAergic neurons and inhibits substantia nigra pars compacta

• contain D1 neurons form direct pathway

direct pathway of basal ganglia

• descneding inputs from cortex and D1R input to striatum activates MSN which disinhibit the thalamic neurons through the globus pallidus internal

• MSN contains D1R, GPCR and it is exitatory and causes greater depolarization of membrane potential, this would facilitate intended motor output

• direct pathway activates intended motor behaviour