Neurotransmission and Modulation Flashcards
What is the Nerst Equation?
RTln([ionint])/zf ln([ionext])
Describe the structure of pentameric ligand gated ion channels (give examples):
- 5 subunits (2α,β, γ, δ)
- Each subunit contains 4 sequences (M1-4)
- M2 region is a pore loop where ligand can attach
Examples: nAChR (excitation); GABAa (inhibition); GlyR (inhibition)
What is the structure of the nAChR channel?
- Pentameric ligand gated ion channel
- 2 ACh bind to pore loops (on M2 sequence)
- Allows the passage of cations (Na+, K+, Ca2+)
- Causes depolarisation and excitation
Name tetrameric ligand gated ion channels and briefly describe their structure.
- Mainly glutamate receptive
Examples: AMPA; Kainate (GluK); NMDA
What is the structure of the Nav channel and why is it significant during depolarisation?
- 4 subunits each with sequences S1-6
- Pore loop between S5-6 which forms channel
- S4 is voltage sensitive
Significance:
- Probability of one domain being open is m
- Probability of voltage inactivated domain being open is h
- Therefore total probability is m^3h
- Probability of opening increases with depolarisation
Assumes all the domains are independent (big assumption)
How was the K+ channel discovered?
- ‘Shaker’ mutant in Drosophila acted same as WT exposed to K channel inhibitor
- Implied channel responsible for K+
Describe current-voltage experiments by Hodgkin/Huxley
Transient inward and delayed outward current seen
Ion substitution experiments:
- Determined Na+ caused inward current and resulted in depolarisation
- Determined K+ caused outward current
Channel blocking experiments
- Tetrodoxin blocks Nav channel
- No action potential then stimulated
What is saltatory conduction and how does it work?
Depolarisation jumps between nodes leading to faster conduction and lower metabolic load.
Mechanism:
- Raises capacitance due to myelination
- Oligodendrocytes in CNS and Schwann cells in PNS
- Creates elongated local currents
What is the purpose of the hyperpolarisation?
- Prevents antidromic (backwards) travel
- Increases re-activation rate of deactivated Nav channels
- Therefore shortens the absolute and relative refractory periods
- Reduces risk of overstimulation (e.g. epilepsy)
How do action potentials code given they are all-or-none?
- Spike frequency coding (larger current = higher frequency)
- Pattern of APs (regular/irregular/intrinsic bursts)
- Width of AP (depending on temperature and kinetics (e.g. KIR slower vs. type A K+ channels)
- Can be modulated by other chemicals
- Repolarisation can be accelerated/decelerated (e.g. by Ca2+ channel activation)
What is passive linear integration? (give examples)
The combination of all incoming signals in a dendrite, changing the probability of progagating an AP>
Examples:
- EPSPs
- IPSPs
- Morphology (how many synapsing dendrites and amount of branching)
- Shunting inhibition
What is shunting inhibition? What is its use?
- Opening Cl- channels
- Since Ecl is close to resting potential this does not change membrane potential significantly
- Decreases change of AP propagating
Use: filters out signal noise since excitatory currents effectively divided by amount of inhibitory input.
Which receptors and molecules result in EPSP and IPSP production?
EPSPs: glutaminergic synapses (distal part of dendrites)
- AMPA receptors are rapid
- NMDA type receptors and slower and can be Mg2+ dependent
- Both have intrinsic Na+/Ca2+ channels causing excitatory currents
- Kainite receptors e.g. GLUK4
IPSPs:
- GABAergic or glycinergic at proximal dendrite sites
- Have integral Cl- channel repolarising the cell
What is active integration and what evidence supports it?
Dendrites propagating/initiating an AP.
Evidence: simultaneous patch-clamp recordings in soma and dendrites
Where can back propagation occur?
- When Nav1.2 channels present (do not inactivate)
- For spike timing dependent plasticity.
Contrast neurotransmitters and neuromodulators:
Time course of action: NT much faster (ms rather than mins/hrs)
Release-effect coupling: NTs strongly coupled to effect; NMs have 2nd messengers which outlast their release
Information route: NT = specific (one cell to next); NMs = non specific populations
What is a ‘brain state’ and how might it be achieved using neuromodulators?
Where activity of nearly all brain altered e.g. asleep or awake state
Using modulators such as DA, NA, SHT, HA which amplify their signals using GPCRs (since only a few cells secrete them).
Describe the structure of an electrical gap junction synapse:
- Tight electrical coupling
- Each cell membrane has hemi-channels of connexins which are aligned
- Tetra membrane spanning proteins dock hemichannels in place
- Synchronises electrical activity so common in interneurons of neocortex
- Transmission occurs in both directions
How was vesicular release discovered?
- Mepps recorded post-synaptically
- Always in quanta of a similar size
- Suggested vesicular release
What roles do glial cells have? (5 points)
- Provide structural support
- Take up neurotransmitter to stop stimulation.
- Spatial buffering of K+
- Metabolic support e.g. astrocyte foot processes which provide lactate for respiration
- Glutamatergic signalling support
- Control of channels using gliotransmitters
Give an example of a gliotransmitter and describe its mechanism of action:
- L-serine converted to D-serine
- By enzyme racemase in glial cells
- D-serine acts as a co-agonist for NMDA receptors
- Increasing long term potentiation
Detail the different categories of neurotransmitter and give an example of each:
Neuropeptide (8-30 aas):
- Orexins and hypocretins for wakefulness
Small molecules (catecholamines):
- DA, SHT, HA, NA, ACh
Amino acids:
- GABA, glycine, glutamate, asparagine
Gaseous:
- CO, NO
- Not vesicularly contained as can freely diffuse
Where is dopamine produced and what is its main function?
- Substantia nigra
- For voluntary movement and reward
- Depletes in Parkinson’s disease
Where is serotonin produced and what is its main function?
- Raphe nucleus
- Affects sleep-wake cycle and mood
Where is histamine produced and what is its main function?
- Hypothalamus
- For arousal and energy/metabolism
How is glutamate synthesised? How is ACh synthesised?
Glutamate:
- From α-ketoglutarate or glutamine
- Either in astrocytes or pre-synaptic terminals.
ACh:
- From acetyl CoA by choline acetyltransferase (CAT)
- In mitochondria
Detail how the Ca2+ sensitive release mechanism works:
- Vesicular synaptobrevin spontaneously twists together with terminal SNAP-25 and syntaxin
- Synaptotagmin blocks complete zipping of SNAREs until Ca2+ bound
- Fusion pore formed, releasing contents.
How are vesicles recycled?
Kiss-and-run release:
- Partial emptying
- Vesicle engulfed back into cell for recycling
Clathrin dependent:
- Vesicle is clathrin associated
- Clathrin re-forms vesicle after combination with cell membrane
Suggest ways in which activity at a synapse is stopped and reset after stimulation:
Remove neurotransmitter from cleft:
- Diffusion
- Take into glial cells
- Breakdown
Stop vesicle release:
- Close/inactivate Ca2+ channels
- Deplete vesicle number
Recycle vesicles:
- Clathrin dependent
- Kiss-and-run
Post-synaptic control:
- Stopping secondary messengers
Describe the different GABA receptors:
GABAa = Ionotropic
- Penatmeric family with Cl- channel
GABAb = metabotropic
- Made from an obligate heterodimer of GABAb1 and GABAb2
- Inhibited by GABA reuptake
How do naive GABAa differ from mature GABAa receptors?
- Naïve GABAA channels start excitatory
- Become inhibitory as expression of KCC2 co-transporter increases
- Reduces intracellular [Cl-]
How are GPCRs activated?
- GDP is displaced and replaced by GTP from α-subunit which dissociates.
- Conformational change of TM segments 3 and 6 where ‘ionic lock’ is broken
How are GPCRs inactivated?
- GTP cleaved (in time) by intrinsic GTPase activity to inactivate
- Β-arrestin binding of GTP bound GPCR results in endocytosis of receptor (can later be reinstated or recycled by acidification of vesicle)
How can the spatial-temporal landscape be changed during signalling plasticity?
- Receptor desensitisation (e.g. phosphorylation)
- Signalling pathway type (e.g. same receptor attached to Gs or Gi)
- Density and type of receptor (e.g. receptor internalisation)
- Gene expression change.
What causes Myasthenia Gravis? How can it be treated?
Autoimmune disease caused by antibodies against nAChR
- Causes problems at NMJ so weakened muscle movement
Treatment;
- Increased ACh concentration by using AChE inhibitor
What causes Parkinson’s disease?
Progressive autoimmune disorder of movement with difficulty in initiating and stopping voluntary movements:
- Due to loss of dopamine neurones of the substantia-nigra (SubNR) of the basal ganglia.
- Causes imbalance between indirect and direct pathways of the striatum meaning motor cortex is inhibited.
- Since dopamine reduction causes both reduced stimulation (via D1R receptors) and increased inhibition of movement (via D2R receptors)
Describe the direct pathway to stimulate the motor cortex:
- Cortex excites striatum
- Increases inhibition of substantia nigra by striatum
- Therefore SubNR reduces inhibitory signals to thalamus
- Increased movement
Direct pathway is promoted by dopamine binding D1R receptors in striatum
Describe the indirect pathway to inhibit the motor cortex:
- Cortex excites striatum
- Increased inhibition of globus Pallidus
- GP inhibition leads to excitation of subthalamic nucleus and SubNR
- SubNR produces inhibitory signals
- Reduces stimulation of thalamus and hence reduced movement.
Dopamine reduces inhibition of movement by binding to D2R receptors in striatum:
- Dopamine (D2R) inhibits striatal neurons
- GP increases inhibitory signals
- Subthalamic nucleus reduces glutamate (excitatory) release
- SubNR produces less GABA (inhibitory)
- Thalamus more excited hence MORE movement.
What are different treatment options to increase Dopamine levels?
- Increase dopamine concentration: e.g. use L-dopa (a precursor)
- Block dopamine degradation: MAO inhibitors
- Stimulate dopamine receptors using dopamine mimicry
- Block reuptake of dopamine (cocaine!)
How do glial cells influence signalling at a synapse?
Modulation of neurotransmitters:
- Recycling and reuptake (ACh; glutamate-glutamine cycle)
- Reduces metabolic cost and increases speed of recovery
Modulation of environment of synapse:
- Ion homeostasis: must be balanced to allow depolarisation but prevent overexcitability
- Keeps K+ conc low extracellularly
- pH: affects receptor conductance
Synaptic plasticity:
- LTP/LTD: gliotransmitters e.g. D-serine released increases activity of NMDA receptor
- Formation of new synapses and pruning of old.
How can synaptic efficacy be changed? [Equation]
Total effect of neuron A on B = NPQ
N = number of release sites
P = probability of release
Q = postsynaptic effect of release
Detonator synapses can stimulate post-synaptic AP on their own (Integrative synapses cannot).
Describe the mechanisms behind short term potentiation:
Frequency dependent short-term plasticity:
- Facilitation (≈50-300ms) (may be due to Ca2+ influx)
- Augmentation (≈7s)
- Post-tetanic potentiation (1 min)
Paired-pulse facilitation: synaptic response to second pair of stimuli is greater (≈300ms)
- Tetanic is due to synaptic modification aiding neurotransmitter release (E.g. residual calcium)
- Post-tetanic potentiation (PTP) – due to protein kinase C activation (increases sensitivity to Ca2+)
What are the mechanisms of synaptic depression?
Decrease in response following repeated stimulation:
- >30Hz is due to vesicle depletion from active zone
- <30Hz is due to inactivation of presynaptic Ca2+ current
What are the mechanisms of long term potentiation?
Spike timing dependent plasticity:
- Causal (post-synaptic spike leading to pre-synaptic spike = LTP)/acausal relationship (post-synaptic spike proceeding pre results in LTD (dampens EPSP)
- Effectively gives ‘coincidence detection’ property of synapses as depolarisation occurs to displace Mg2+ and synaptic stimulation via glutamate occurs to activate NMDA
Frequency dependence:
- Tetanisation of input fibres causes LTP in that synapse but LTD in adjacent synapses
What are the differences between NMDA and AMPA receptors?
NMDA receptors = coincidence detectors (require both depolarisation and glutamate) and allow Na+ AND Ca2+ current. AMPA only require glutamate and allow Na+
How does the [AMPA receptors] affect synaptic efficacy?
More AMPA = increased efficacy (LTP involves insertion of AMPA)
Presynaptic:
- Unmuting of silent pre-synapses by AMPA addition (previously only NMDA present (blocked by Mg2+ usually))
- AMPA insertion matures synapse by increasing facilitation of future activation and reduction in propagation failure rate
- Reduction of whispering synapses (where glutamate concentration in vesicles too low to stimulate AMPA)
Post-synaptic – increase in responsiveness (no. of receptors or single channel conductance)
- Reduction in ‘deaf’ silent synapses by AMPA insertion
How is synaptic efficacy measured?
Measured by slope of EPSP:
- Index of synaptic efficacy (internal excitatory current (EPSC) much faster than the potential induced)
- EPSC altered by receptor density, properties and state (active?)
What is Hebbian plasticity? What are the three properties which change the rate of its induction?
Hebb: “when an axon of cell A is near enough to excite cell B and repeatedly takes part in firing some growth/metabolic changes take place in one or both cells to increase efficiency of A firing B”
Induction depends on;
- Cooperativity: need to stimulate multiple afferent to induce LTP
- Input specificity: only certain target synapses will cause LTP when stimulated individually
- Associativity: weak and strong inputs tetanised together both show LTP
What are the mechanisms behind cooperativity and input specificity?
Co-operativity = need to stimulate multiple afferents to induce LTP:
- Stimulus must be prolonged to overcome GABAergic inhibition of post-synapse
- Overcoming resistance is difference between short and long term potentiation
Input specificity = only certain target synapses will cause LTP when stimulated individually
- Synaptic tagging of tetanised synapses allows protein synthesis in general cell body to affect only specific synapses stimulated
- Opposing theory is local protein synthesis around stimulated synapse
Give examples which demonstrate the impact of neural context on long term potentiation:
Cortical/hippocampal network:
- Excitation leads to long term potentiation
- Exocytosis of AMPA
Cerebellar response:
- In cerebellum error signal drives synaptic depression (goal is to REDUCE excitation)
- Responsible for vestibulo-Ocular reflex: retinal slip induces climbing fibre activation (mossy fibre feedback of movement from vestibular organ does not match eye movement causing retinal slip which is the error signal)
Detail the mechanism behind cerebellar plasticity
Excitation leads to long term depression (goal is to REDUCE excitation):
1. Climbing fibres activate AMPA receptors with complex spike (Ca2+ influx with Na+ spike overlayed)
2. When activated together – parallel fibre via metabotropic glutamate receptors and climbing fibre causing Ca2+ influx
3. Activates protein kinase C –> DAG + PLC
4. Endocytosis of AMPA
(Responsible for vestibulo-Ocular reflex)
What experimentation demonstrates the frequency dependence of Hebbian plasticity?
Shown by low frequency stimulation (low 1Hz) of homosynaptic Schaffner collateral (in CA1 of hippocampus) leading to homosynaptic LTD.
How did experimentation show the existence of silent synapses?
Before LTP:
- Synaptic currents at +55mv but not -65mv (glutamate activating NMDA but not AMPA)
- LTP causes appearance of AMPA currents.
- Long term changes require protein synthesis (shown as protein blocker stops LTP (but not STP))
How was NMDA participation in spike timing dependent plasticity (STDP) determined?
Using NMDA receptor agonists (AP5):
- Removes response (molecular coincidence detection role)
- Both detect pre-synaptic glutamate release (back propagation) and postsynaptic depolarisation
Using MK801 (blocks NMDA pore itself not glutamate binding site):
- Only stops LTD when introduced pre-synaptically (not post)
- E.g. Ocular dominance plasticity in visual cortex (mechanisms like LTP in CA1 hippocampus) blocked by NMDA agonist
What is the BCM rule? What are the mechanisms behind it?
Plasticity is frequency dependent and HFS input shifts threshold so higher frequencies required to cause further LTP.
Stabilises firing rate between neurons (since plasticity is relative not absolute)
Achieved by:
- Different receptor densities
- Different receptor subtypes (due to different subunit composition)
- E.g. intracellular GluN2 subunits of NMDA receptor
GluN2A > GluN2B = shifts curve right = LTP (mature synapse) but higher threshold
GluN2B > GluN2A = shifts curve left = LTD (immature synapse)
Describe experimentation which proves LTP can occur without STP?
Causal events lead to strengthening of synapse (see STDP):
- Short-term facilitation: 5-HT induced via CYP-sensitive receptor
- Long-term facilitation: also 5-HT induced by a distinct mechanism via CYP-insensitive receptor – can experience LTP without STP
- Shown by blocking STP using CTP (LTP still occurs)
What do the Morris water maze experiments demonstrate about memory dependence on LTP in the Hippocampus?
Use rodents swimming in water to find a platform (Morris water maze task) – learn to expect a platform in a specific corner of pool.
Blocking experiments:
- Memory of platform place no longer shown if NMDA receptors blocked (stops LTP)
- Memory not shown if CA1 restricted NMDAR1 knockout mouse
- CaMKII knock-out mouse (more specific than NR1) – again showed no preference
Saturation experiments:
Correlation: inhibitory avoidance training (in CA1)
- Used electrodes to measure LTP
- Harder to induce further potentiation in these neurons when stimulated further (compared to control); suggests already stimulated (BCM rule)
How do amygdala fear experiments suggest LTP is involved in memory formation?
Auditory stimulus paired with an electric shock – induces behavioural ‘freeze’ when sound heard (requires LTP):
- Plasticity linked to memory (hippocampal and amygdala connections)
- Tag AMPA receptor with GluA1 C-tail (injected using a virus) which reduces efficacy
- Led to significant reduction of fear response after training (less LTP)
Using different pitches of sound (one came with shock (= CS+); one did not (+CS-)
- Sound coupled with shock induced increase AMPA:NMDA ratio in synapses
Erasure of fear memory:
- Using optical stimulation to induce LTD resulted in removal of memory (reversal of LTP)
List experimental evidence for NMDA/AMPA involvement in LTP:
- Amygdala fear conditioning experiment with AMPA receptor density measurement (tag with GluA1 C-tail)
- Morris water maze experiments (blocking/knockout mice for NMDAR or CAMKII)
