Block 1: the neuron

Question 1

Ouline the key step of neurotransmission. Detail the potential drug targets within this process.

Answer 1

1) Uptake of precursor molecules – these are taken up to be converted to neurotransmitters in their active form (precursor transporter can be targeted)
2) Synthesis – enzymes are used to convert precursors to active neurotransmitters (these enzymes can be targeted)
3) Packaging into vesicles – involves a transporter on vesicular membrane
4) Breakdown of excess transmitter – if vesicles are filled to capacity (involving another enzyme which can be targeted)
5) Action potential – involves an ion channel which can be targeted (voltage gated sodium and potassium channels)
6) Pre-synaptic calcium entry (another ion channel)
7) Neurotransmitter release – pre-synaptic vesicles fuse with membrane to release contents into the synaptic cleft
8) Crossing the synaptic cleft
9) Binding to postsynaptic cell – involves a post-synaptic receptor
10) Breakdown of neurotransmitter in synaptic cleft – involves another enzyme
11) Re-uptake into presynaptic terminal – involves a transporter
12) Can alternatively be taken up into adjacent cells such as microglia – involving a transporter
13) Other receptors are expressed on the pre-synaptic terminal – these are for negative feedback control, and these receptors can also be targetted

Question 2

Compare and contrast affinity, efficacy, potency, and occupancy regarding drugs.

Answer 2

Affinity is a measure of the strength of association between a ligand and receptor (governed by the rate of associate and dissociation)- low affinity molecules will dissociate quickly.
Efficacy is a measure of a drug’s ability to evoke a response when bound to its receptor- drugs with low efficacy have low activation rates and high deactivation rates, and will evoke low cellular responses compared to those with higher efficacy (binding=affinity; activation=efficacy).
Potency is similar to efficacy, but refers to the concentration at which a drug elicits a given response, rather than the maximal response which can be produced (there is no unit for potency, it is a comparative measure). Two drugs can have the same efficacy, but one with a greater potency will elicit its maximal response at a lower concentration than the other.
Occupancy refers to the proportion of receptor sites which are occupied by a particular ligand (typically increases with drug concentration in a hyperbolic manner).

Question 3

What is EC50, and how can it be identified?

Answer 3

EC50 (effective concentration 50) is the concentration of agonist which elicits a half-maximal response- it i the relationship betweend drug concetration and efficacy. This can be easily identified by converting drug concentration on a dose-response curve to a log scale, giving a sigmoidal dose-response.

Question 4

Explain the two-state model of receptor activation with reference to the actions of agonists and antagonists.

Answer 4

The two-state model states that receptors can exist in either a resting state or an activated state, expressed as R and R, respectively. This is a dynamics relationship, and receptors fluctuate from one state to the other all the time. In the absence of an agonist, the equilibrium will lie to the resting state, but will shift when an agonist binds. The greater affinity an agonist has for the R state, the greater its efficacy. Antagonists have equal affinity for both states, and so have no effect on equilibrium, however they affect the ability of other drugs or endogenous ligands to bind to either R or R*, preventing equilibrium shifts.

Question 5

Explain what is meant by a “constitutively active” receptor with reference to the two state model.

Answer 5

If a receptor is “constitutively active”, it means that in its resting state (under normal physiological conditions), it continuously fires action potentials, without the need for agonist binding. Agonists to these receptors will still preferentially bind to the R* state, and shift the equilibrium further toward this active state. However, there are also inverse agonists, which preferentially bind to the R state, and will therefore deactivate the receptors. Antagonists will block the effects of agonists and inverse agonists equally, in a dose-dependent manner.

Question 6

Explain actions of a full agonist, partial agonist, antagonist, partial inverse agonist, and full inverse agonist on a constitutively active receptor with reference to the two state model.

Answer 6

A) Full agonist – a full agonist acting on constitutively active receptors will shift the equilibrium toward the active state, resulting in more activity and a maximal response at sufficient concentrations.
B) Partial agonist – a partial agonist acting on a constitutively active receptor will also shift the equilibrium toward the active state, resulting in greater activity. Although this increases the response, it will never elicit a maximal output due to their lower efficacy.
C) Antagonist – Since antagonists have equal affinity for both the resting (inactive, “R”) and active (“R*”) states, an antagonist alone acting on many receptors will elicit no overall change in response, and are thus said to be “silent”.
D) Partial inverse agonist – a partial inverse agonist acting on a constitutively active receptor will reduce the response by shifting the equilibrium towards the inactivated (“R”) state. It will not completely silence the response due to sub-maximal efficacy.
E) Full inverse agonist – a full inverse agonist acting on a constitutively active receptor will also reduce the response by shifting the equilibrium towards the inactivated state, and will completely deplete the response at sufficient concentrations.

Question 7

Define antagonists, including how they differ from inhibitors, and the differences between competitive and non-competitive antagonists.

Answer 7

Antagonists bind to receptors but do not activate them- they possess affinity but lack efficacy. Inhibitors block enzymes and transporters, whereas antagonists block receptors. Competitive antagonists compete with agonists and endogenous ligands at the same binding site (higher affinity and higher concentration will win out). Non-competitive antagonists block the effects of agonists from different binding sites, causing conformational changes which reduce the agonist’s affinity to its binding site. Some competitive antagonists bind covalently (irreversibly) to agonist binding sites, meaning that agonists cannot displace the antagonist (agonist effects are then dictated by occupancy of the antagonist rather than competition for affinity).

Question 8

Describe how competitive and non-competitive antagonists affect the sigmoidal dose-response curves of agonists.

Answer 8

In the presence of a competitive antagonist, more agonist is required to elicit the same response as if there were no antagonist (i.e. graph shifts to the right, as if it had a lower potency, but efficacy has not been affected).
In the presence of a non-competitive antagonist, marginally more agonist is required, but additionally, peak response is dramatically reduced (i.e. graph is marginally shifted to the right as though it has lower potency, but it is also dramatically shrunk down such that %max response is reduced as though the agonist had lower efficacy).

Question 9

Describe the structure and function of VGICs.

Answer 9

VGICs are composed of four separate domains arranged around a central pore, each of which has six transmembrane segments (amino acid sequence crossing cell membrane six times above/below). Segment 4 is the voltage sensor, and is largely comprised of positively charged amino acids; segments 5 and 6 of each domain line the channel pore.
VGICs are closed at resting membrane potential, but rapidly open and close in response to changes in membrane potential (transiently). Their main purpose is to conduct APs involved in depolarisation and repolarisation of the cell membrane. They cycle through 3 conformational states; resting (closed), activation (open), and inactivated- this results in a two-step mechanism of inactivation (ball-and-chain block followed by pore closing). The time the channel takes to completely inactivate is known as its refractory period. It cannot be reopened until it is fully closed (this governs the frequency at which neuronal signals can fire).

Question 10

Describe the general trends in structure and function of the LGICs.

Answer 10

For all LGICs, binding of a ligand to the orthosteric site triggers conformational change resulting in the conducting (open) receptor state. Modulation can occur by the binding of endogenous or exogenous modulators to allosteric sites. LGICs mediate fast synaptic transmission (milliseconds), in the nervous system. They are comprised of many independent subunits which form a receptor complex, and these are assembled around a central ion pore (heteromultimers). This makes LGICs highly variable, and there are numerous possible expressions for every receptor complex (for instance, the GABA-A receptor requires 5 subunits in order to function, but there are 19 different potential subunits which can be transcriped in the genome). Specific subunit compositions confers specific biophysical properties of receptor complexes, and heterogeneity of receptors across different areas of the nervous system allows us to target specific receptor compositions with pharmacology.

Question 11

Describe the structural and functional characteristics of the 3 classes of LGICs: Cys-loop receptors, ionotropic glutamate receptors, and P2X receptors.

Answer 11

Cys-loop receptors are pentamers (require 5 subunits), glutamate receptors are tetramers, and P2X receptors are trimers.
Cys-loop receptors (e.g. Nicotinic Ach receptor, GABA-A receptor, glycine receptors, etc.) are so named for their “loop” in the extracellular (N-terminus) domain where two cystine residues are connected by a disulphide bridge. They are usually comprised of two alpha-subunits + 3 others, and each subunit has four transmembrane segments (M1-4). The pore is lined by transmembrane segment 2 helices.
Ionotropic glutamate receptors (AMPA, kainate, and NMDA receptors) have four subunits with shortened M2 domains, as well as an intracellular C-terminus. Their conformation is considered to be a “dimer of dimers” as they consist of two pairs of the same subunits.
P2X receptors are ATP-gated channels.

Question 12

Describe the typical structure of GPCRs and their main areas of variation.

Answer 12

The typical 2D structure of a GPCR consists of a common core domain of 7 transmembrane alpha-helices, an extracellular N-terminus and intracellular C-terminus, with helices connected by 3 intracellular and 3 extracellular loops. The length of the N-terminus, and the location of the agonist binding domain are the main areas of variation between different GPCRs.

Question 13

Describe the stucture and function of rhodopsin-like GPCRs.

Answer 13

Rhodopsin-like GPCRs are involved in phototransduction in the visual system which bind small molecule agonists (e.g. ammine neurotransmitters, neuropeptides, purines, etc). They are characterised by a short N-terminus; the seven helices form a cavity where the ligand binding site is typically found; they have cysteine residues in extracellular loops 1 and 2 which form a disulphide link to provide stability (and 3D structure packing); proline residues in helices 6 and 7 introduce kinks into the alpha-helices to facilitate conformational change due to agonist binding; and aspargine residue in transmembrane 2, as well as a triplet of residues (AspArgTyr- AKA the “DRY” motif) in the second intracellular loop are important for receptor activation.

Question 14

Describe the stucture and function of secretin-like GPCRs.

Answer 14

Secretin-like GPCRs are activated by short peptide agonists (e.g. glucagon, GLP-1, calcitonin, etc). Their structure is stabilised by 3-sulpide bonds within 6 highly conserved Cys residues. They have a very long N-terminus tail (100-160 residues) which incorporate part of the ligand-binding domain.

Question 15

Describe the stucture and function of metabotropic glutamate GPCRs.

Answer 15

Metabotropic glutamate receptors are an example of class C GPCRs (along with GABA-B receptors and calcium-sensing receptors). They have an extremely large N-terminus with a binding domain known as the “venus fly-trap” module. They commonly have allosteric binding sites as well within 7TM domains. Class C GPCRs form dimers to form a signalling complex (homodimer=dimer of the same receptor subtype; heterodimer=dimer of different receptor subtypes).

Question 16

Describe the functional mechanism of GPCRs.

Answer 16

GPCR signal transduction can be broken down into three steps: receptor, G-protein, effector. The G-protein is the secondary messenger in the system (stands for guanine nucleotide-binding regulatory protein), and these are heterotrimeric (consist of an alpha, beta, and gamma subunit). Alpha-subunits possess GTPase activity, and can bind to either GDP or GTP. Binding of GDP is associated with the inactive state of the G-protein, binding of GTP is associated with the active state.

1) Upon agonist binding, the receptor attracts the GDP bound G-protein (basal state)
2) The receptor bound G-protein then releases GDP from the alpha-subunit, allowing GTP to bind
3) The G-protein then dissociates into the alpha subunit bound to GTP (GTP-a)and a beta-gamma dimer (By-complex). The agonist also dissociates from the receptor at this point
4) The GTP-a and the By-complex can each then bind to effector proteins to modulate downstream signalling
5) Intrinsic GTPase on the alpha subunit hydrolyses the GTP to form GDP, causing the GTP-a and By- complexes to dissociate from their effector proteins (inactivating them)
6) The G-protein trimer then reforms (goes back into basal state)

Question 17

Describe the conformational change associated with agonist binding of a GPCR.

Answer 17

The binding of the agonist to the GPCR causes a conformational change – transmembrane (TM) domains 3 and 6 move away from each other, and TM6 also rotates 30 degrees. This opens up a cleft where the C-terminus of the G-protein alpha-subunit can bind, and is then activated by interaction with the DRY motif, as well as other sequences.

Question 18

What are the main functions of RTKs and what sets them aside from LGICs and GPCRs?

Answer 18

RTKs (receptor tyrosine kinases) have very different structures from LGICs and GPCRs- they are very large single unit proteins (up to 1000 residues) with a single membrane-spanning helical region (structure and size of extracellular and intracellular domains varies greatly). They play a major role in controlling cell division, growth, differentiation, inflammation, tissue repair, apopotosis, and immune responses (mutations of these proteins is linked to cancer). They control the actions of a wide variety of protein mediators, including growth factors, cytokines, and hormones such as insulin and leptin. Activation of RTKs is usually associated with long-term changes in cell function, where gene expression is required.

Question 19

Describe the structure of nuclear receptors and briefly their mechanism of action.

Answer 19

Nuclear receptors are very large proteins which consist of an N-terminus regulatory domain (highly variable sequence); a DNA-binding domain (high conserved- binds to DNA using specific sequences called hormone response elements); a hinge region (flexible domain which connects the DBD and LBD and influences trafficking and subcellular distribution); a ligand-binding domain (moderately conserved in sequence but highly conserved in structure); and a C-terminus domain (highly variable). The hinge region allows for conformational changes upon ligand binding, and the complex moves to the nucleus, binding with DNA via the DNA-binding domain. Subsequent association with accessory proteins (e.g. co-activators and co-repressors) results in gene transcription being switched on/off.

Question 20

Compare and contrast type I and type II nuclear receptors.

Answer 20

Type I nuclear receptors are for steroid hormones (glucocorticoid and mineralocorticoid receptors, oestrogen, progesterone, and androgen receptors). Activation of type I receptors often causes a negative feedback effect. They are typically complexed with heat shock proteins which anchor them to the cytoskeleton and dissociate upon ligand binding, at which point they form homodimers which translocate to the nucleus.

Type II nuclear receptors have ligands which are already present within the cell (under normal circumstances, these ligands are bound to a deactivating protein complex from which they will dissociate in order to become active and translocate to the nucleus via pores where the receptors are found). Example include peroxisome proliferator-activated receptor, liver oxysterol receptor, and thyroid receptors. type II receptors typically operate as heterodimers alongside the retinoid receptor (RXR) to mediate positive feedback effects (amplify biological events).

Question 21

Describe the actions of transcription co-regulator proteins.

Answer 21

Once nuclear receptors are bound to their hormone response elements, they can recruit other proteins called transcription co-regulators. These facilitate or inhibit the transcription of the associated target gene into mRNA. Nuclear receptors may bind specifically to a number of coregulator proteins, thereby influencing what happens within the cell. Binding of an agonist to its nuclear receptor induces a conformation that preferentially binds co-activator proteins to promote gene transcription. Binding of an antagonist induces a conformation of the receptor that preferentially binds to co-repressor proteins which repress gene transcription.

Question 22

Describe the structure and function of the three main neuronal cytoskeletal components.

Answer 22

1) Microtubules are the largest component in neurons- they are composed of alternating strangs of alpha and beta-tubulin to form a long, hollow tube. These two tubulins for a heterodimer, and these heterodimers combine to form a protofilament. Roughly 13 of these protofilaments are arranged linearly to give a tube structure. They are also polarised, and are highly dynamic (positive end can grow, shrink, or be stable). They can also exist in branches (protofilaments break apart and new filaments are formed to produce 13-filament units)
2) Microfilaments are the thinnest fibres of the cytoskeleton (5nm diameter). Each filament is composed of two strands of actin polymers (two G-actin monomers form an F-actin polymer). These are found in abundance in neurites, and are also polarised with a growing end and a stable end. Growing of microfilaments is an active process- whenever a G-actin monomer is added to the end of a filament, ATP is hydrolysed for energy. Microfilaments are typically anchored to the neuronal membrane by actin-binding proteins. They provide stability, but primarily confer motility to the neuron.
3) Neurofilaments are intermediate in size (10nm diameter). They are very strong and stable, and as such are referred to as the ‘bones’ of the cytoskeleton. Neurofilament monomers pair up and coil to form dimers. Two of these dimers combine to form protofilaments. Two of these protofilaments combine and coil to form protofibrils. Three of these protofibrils twist around each other to form the neurofilaments (each filaments composed of 24 monomers). They are a common biomarker in identification of neurodegenerative diseases (for instance, neurofibrillary tangles in AD).

Question 23

Explain the process of axoplasmic transport. Compare anterograde vs retrograde and fast vs slow.

Answer 23

Axoplasmic transport is the movement of proteins along the axon of a neuron. Axons lack ribosomes, and therefore cannot synthesise proteins – any proteins within the axon must be synthesised in the soma and transported to the axon. Microtubules act as the “tracks” for transportation. Axoplasmic transport can be fast (50-400mm/day) or slow (<8mm/day), and can be forward (anterograde) or backward (retrograde). Retrograde axoplasmic transport is necessary as there are certain proteins which are taken up at the axon terminals (e.g. growth factors which need to be transported to the soma).

Anterograde transport involves kinesins- motor proteins which carry protein-filled vesicles along the microtubule from the -end to the +end (this process is active – for every step the kinesin takes, 1 molecule of ATP is hydrolysed). - Kinesin has a structure of two feet which “walk” along the microtubule, a body, and a head with two arms which grip the cargo

Retrograde transport involves dyneins – these motor proteins also “walk” along microtubules while carrying protein-filled vesicles as cargo, this time from the +end to the -end. Dynein has a similar structure to kinesin, and also requires hydrolysis of 1ATP per step taken

Fast axonal transport is bidirectional – it can be anterograde or retrograde (typically can transport membranous organelles such as mitochondria, secretory vesicles, large dense core vesicles, and smooth ER) – it uses these motor proteins to actively transport cargo. Slow axonal transport does not use motor proteins – it is responsible for the movement of cytosolic and cytoskeletal proteins (e.g. adding more length to a microtubule) via sliding and polymerisation. It is unidirectional (anterograde only). Slow transport can be characterised as a or b – with SCa being microtubules and neurofilaments (0.1-1mm/day), and SCb being actin and cytosolic proteins (2-4mm/day).

Question 24

Explain how the resting membrane potential of -70mV is established and maintained.

Answer 24

Concentration (chemical) gradients of a solute mean that it will diffuse from areas of high to areas of low concentration. Charge (electrical) gradients mean that positively charged solutes will move to more negative areas, and vice versa. A single charged solute reaches electrochemical equilibrium when its concentration and charge gradients are equal and opposite. This does not mean that the solute has equal concentrations or charge on either side of the membrane. The value of the potential difference at electrochemical equilibrium of a solute is defined as its equilibrium potential.
At resting state, the membrane is slightly permeable to Na+, but it is around 25-30 times as permeable to K+, due to the presence of potassium leak channels. K+ will therefore freely diffuse down its chemical gradient until it reaches electrochemical equilibrium at -70mV, which is close to its equilibrium potential (-90mV) due its relatively high permeability. By contrast, Na+ will be further from its equilibrium potential of +65mV. This means that at resting membrane potential, the intracellular side of the plasma membrane is 70mV more negative than the extracellular side.
However, some Na+ will still leak into the cell, and some potassium will still leak out of the cell. If this goes unchecked, both the charge and chemical gradients may be joepordised, and the resting potential may dissipate. To correct for this, the sodium-potassium pump (Na+/K+ ATP-ase) maintains resting potential by hydrolysing one molecule of ATP to pump 2K+ in and 3Na+ out of the cell.

Question 25

Describe graded potentials with reference to EPSPs and IPSPs.

Answer 25

Graded potentials are transient, relatively small depolarising or hyperpolarising events which can arise from activity of post-synaptic receptor activation, or by other stimulation (e.g. physical pressure, light, etc). post-synaptic potentials may be excitatory (EPSP) or inhibitory (IPSP), depending on the ion flux generated by the opening of the receptor in question.
When a neurotransmitter binds to a post-synaptic ionotropic receptor, a conformational change allows passage of ions through the membrane. If these are cations moving intracellularly, the membrane will depolarise (become less negative intracellularly relative to extracellularly); if they are anions moving intracellularly or cations moving extracellularly, the membrane will hyperpolarise. However, the ligand will rapidly dissociate from the receptor, and the channel will shut. The resulting graded potentials arising from transient ion flux will spread outward by local current flow, reducing decrementally (amplitude of depolarisation is greatest at its point of origin). This is because the electrotonic potential will passively propagate in all directions, including back out of the cell through leakage.

Question 26

Explain the principal of summation.

Answer 26

Graded potentials can summate temporally or spatially. This means that either the same post-synaptic density depolarises (in the case of EPSPs) repeatedly in quick succession, or adjacent post-synaptic densities depolarise simultaneously. The summation of many small EPSPs causes cumulative depolarisation of large areas of the neuron.
IPSPs can also contribute to summation, cancelling out EPSPs by hyperpolarising.

Question 27

Describe how an action potential arises.

Answer 27

If summation of EPSPs is sufficient that they reach the axon hillock and cause potential difference to reach threshold (usually -55mV), an action potential (AP) will fire. Voltage-sensing domains of VGNa+Cs the axon initial segment will detect this depolarisation and will open, allowing influx of sodium, causing a large depolarisation event which peaks at around +35mV (though can be as high as +100mV).

Question 28

Explain the propagation of APs down the length of the axon in 1) unmyelinated and 2) myelinated axons.

Answer 28

1) In unmyelinated axons, positive local current flow moves in all directions, including subsequent areas of the axon, causing them to exceed threshold potential and open their VGNa+Cs. Although the current also moves backwards, these areas do not respond due to VGICs being in their refractory period. This results in a “wave” of depolarisation down the length of the axon, with each subsequent section reaching threshold and depolarising while the previous section repolarises due to the closing of VGNa+Cs and the opening of VGK+Cs.
2) In myelinated axons, small nodes (nodes of Ranvier) of VGICs are located between segments of myelinated areas (internode regions). Due to the increased membrane resistance by virtue of the myelin sheath, local currents extend a greater distance faster over internode segments, then stimulate opening of VGICs exclusively expressed in the nodes of Ranvier. This is known as “saltatory” conduction, as the depolarisation “jumps” from one node to the next. It is much faster than continuous conduction, and has the added benefit of reducing metabolic demand of balancing sodium and potassium concentrations down the entire length of the axon with Na+/K+ ATP-ase.

Question 29

Describe the process of synaptic transmission.

Answer 29

When an AP reaches the pre-synaptic terminal, VGCa2+Cs are stimulated to open, and calcium influx results. Intracellular calcium then binds to sensor proteins in the cytoplasm to form complexes which drive the fusion of the vesicles, and subsequent exocytosis of neurotransmitter into the synaptic cleft. After neurotransmitters bind to post-synaptic receptors, they quickly dissociate- at this point, they are rapidly broken down and recycled (to prevent overstimulation). This is usually done by an enzyme within the synaptic cleft, followed by protein carriers.

Question 30

Explain synaptic plasticity with reference to LTP and LTD.

Answer 30

Plasticity is the ability of a synapse to strengthen or weaken over time – it underlies the many physiological and pathophysiological processes such as learning and memory, addiction, and fear conditioning. One of the simplest forms of synaptic plasticity is long-term potentiation (LTP) – this is the ability of a synapse to be strengthened over time. This can happen by increasing the amount of available pre-synaptic neurotransmitter (increasing the probability of neurotransmitter release) or by increasing the amount of post-synaptic receptors (increasing the membranes permeability to ions). - LTP is usually induced by a short, high frequency stimulation of the pre-synaptic neuron (100Hz for one second) – this results in a huge initial increase in EPSP (because you’ve flooded the neuron with signals), which falls back down, but does not reach the original EPSP amplitude – it stays higher (synapse has been strengthened and now has stronger responses to the original signal).
By contrast, long-term depression (LTD) can be induced by low frequency stimulation (LFS – 10-15 mins of 1Hz). - This results in a gradual reduction in amplitude of the EPSPs, which will recover slightly over time but once again will not reach the original EPSP amplitude – the synapse has been weakened.

Synapses also exhibit structural plasticity – as well as the receptor content of the post-synaptic terminal, the shape and structure of synapses can be altered (as well as maturation of synapses, or production of entirely new synapses). Similarly, with LTD, synapses can deteriorate structurally.

Question 31

Describe the process of glutamatergic plasticity in the hippocampus.

Answer 31

The hippocampus uses synaptic plasticity to facilitate learning and memory. It is organised into layers in a tri-synaptic circuit – information comes into the dentate gyrus (DG), which sends signals to the CA3 region then the CA1 region (3-synapse relay). If you stimulate one of these regions, an action potential will be generated, and EPSPs can be recorded on subsequent neurons in the circuit. The main ionotropic, post-synaptic glutamate receptors in the hippocampus are AMPA and NMDA receptors. This is largely mediated by the AMPA receptor, which is activated easily by the binding of glutamate to open its channel. NMDA receptors are generally not active, they require more stimulation in order to be activated (as they have magnesium in the pore which blocks the passage of ions). They are permeable to both sodium and calcium. Large local EPSPs generated by other receptors are required in order to remove the magnesium from the NMDA receptor (such as the aforementioned HFS). Influx of calcium due to NMDA activation starts a cascade of events in the post-synaptic cell (namely by activating kinases which phosphorylate AMPA receptors, affecting their trafficking) – this therefore drives the insertion of more post-synaptic receptors. LFS will also cause calcium influx via NMDA receptors, but at a much lower rate (and smaller amount), which results in the opposite effect due to different downstream pathways. LTP and LTD are not always as straightforward as this, sometimes synapse strength is mediated by different receptor subtypes (which alter conductivity).

Question 32

Highlight the differences in synthesis of small molecule neurotransmitters and large peptide neurotransmitters.

Answer 32

Small molecule neurotransmitters are synthesised through enzymatic interactions with precursor molecules in the pre-synaptic terminal. They are then stored in vesicles. By contrast, large peptide neurotransmitters are synthesised as pro-peptides in the soma alongside enzymes, and are packaged together into vesicles which then migrate to the axon terminal via fast axonal transport.

Question 33

Compare the processes of co-release and co-transmission of neurotransmitters.

Answer 33

Two neurotransmitters can be released from the same pre-synaptic terminal (co-release) or can be released at the same time from different branches of the same axon (co-transmission). These usually involve the pairing of one small molecule neurotransmitter and one neuropeptide. An example of this is at the neuromuscular junction, when acetylcholine is co-released with calcitonin gene-related peptide.

Question 34

Describe the synthesis, transport, and reuptake mechanisms of amino acid neurotransmitters.

Answer 34

Glutamate and GABA are the amino acid neurotransmitters, and are the main excitatory and inhibitory transmitters of the CNS, respectively.

Glutamine is the precursor for glutamate, and is synthesised to its active form in the axon terminal via mitochonrial enzyme glutaminase. Glutamate is then taken up into vesicles via the VGLUT transporter, and held in the axon terminal until release. Once released, it can then be taken up by a neighbouring astrocyte (via EAAT transporter). Within the astrocyte, glutamate is converted back to glutamine by glutamine-synthase. It then leaves the astrocyte and enters the pre-synpatic axon terminal again via the SNAT transporter. Alternatively, it can be directly recycled by entering the pre-synaptic terminal via EAAT transporters (not converted back to glutamine).

Question 35

Describe the synthesis, transport, and reuptake mechanisms of amino acid neurotransmitters.

Answer 35

Glutamate and GABA are the amino acid neurotransmitters, and are the main excitatory and inhibitory transmitters of the CNS, respectively.

Glutamine is the precursor for glutamate, and is synthesised to its active form in the axon terminal via mitochonrial enzyme glutaminase. Glutamate is then taken up into vesicles via the VGLUT transporter, and held in the axon terminal until release. Once released, it can then be taken up by a neighbouring astrocyte (via EAAT transporter). Within the astrocyte, glutamate is converted back to glutamine by glutamine-synthase. It then leaves the astrocyte and enters the pre-synpatic axon terminal again via the SNAT transporter. Alternatively, it can be directly recycled by entering the pre-synaptic terminal via EAAT transporters (not converted back to glutamine).

GABA (gamma-aminobutyric acid) synthesis begins the same way as glutamate- glutaminase converts glutamine to glutamate. However, then another enzyme called glutamic acid decarboxlyase (GAD) converts glutamate to GABA. Additionally, a co-enzyme called pyridoxal phosphate catalyses the process. GABA is taken up into vesicles via VIAAT. Like glutamate, GABA can be taken up directly into the axon terminal via GABA transporter (GAT) or can be taken up by an adjacent astrocyte via GAT, then converted to glutamate by GABA transaminase (GABA-T), and subsequently glutamine by glutamine synthase, which leaves the astrocyte and enters the pre-synaptic terminal via SNAT.

Question 36

Describe the synthesis, transport, and reuptake mechanisms of the monoamine neurotransmitters.

Answer 36

Monoamine neurotransmitters include 5-hydroxytryptamine (5-HT; serotonin), dopamine, noradrenaline, adrenaline, and histamine.

Dopamine is synthesised in the pre-synaptic terminal (as are all biogenic amines). It is a catecholamine. Tyrosine is taken up and converted to L-DOPA by tyrosine hydroxylase (rate limiting step), and this is converted to dopamine by DOPA decarboxylase. Dopamine is then taken up into vesicles by VMAT2 (vesicular monoamine transporter type 2). When released, it can be taken back up into the presynaptic terminal via the dopamine transporter (DAT) to be repackaged (direct recycling) pr can be broken down by mitochondrial enzyme monoamine oxidase. It can also be broken down by cytosolic enzymes in the synaptic cleft, post-synaptic membrane, or elsewhere in the body.

5-HT is an indoleamine. Tryptophan is taken up into presynaptic terminals by the aromatic L-amino acid transporter and then converted to 5-hydroxytryptophan by tryptophan hydroxylase (rate limiting step). This is subsequently converted to 5-HT by aromatic amino acid decarboxlyase. VMAT is used to take 5-HT up into presynaptic vesicles. After release, it can be taken back up into presynaptic terminals via plasma membrane serotonin transporter (SERT) and directly repackaged or can be broken down within the presynaptic terminal by monoamine oxidase.

Histamine is an imidazoleamine. L-histidine is converted to histamine by histamine decarboxylase, which is taken up into vesicles by VMAT2.

Noradrenaline is a catecholamine. Dopamine is taken up into presynaptic adrenergic nerve terminals just as it is in the dopaminergic neurons, but within the vesicles, it is converted to noradrenaline by dopamine beta-hydroxylase (DB-H). Noradrenaline can be taken back up into the terminal via the noradrenaline transporter (NET), or can be repackaged into vesicles or broken down by monoamine oxidase into DOPGAL (or by COMT).
Adrenaline is synthesised from noradrenaline when it leaves the vesicle and is converted in the cytosol by PNMT. The uptake and reuptake mechanism is identical to noradrenaline.

Question 37

What are gap junctions?

Answer 37

Gap junctions are protein structures (pores made from connexin proteins assembled into connexons). These connexons line up to provide a continuous route from the membrane of one cell into the other. There are various connexin protein isoforms. Most of these connexin proteins in heart tissue are concentrated at the end-to-end junctions between myocytes. They are thereby joined by low-resistance pathways (gap junctions) as opposed to chemical synapses – so it works with local circuit currents, but these are continuous from cell to cell.