PCOL3022 - Neuropharmacology

23. Explain the mechanism of action of 5-HT drugs in migraine and with respect to neurogenic inflammation and the trigeminovascular system

1. 5HT2 antagonists directly affect vasculature and reduce activation of receptors in vascular endothelium. 2. Ergotamines are 5HT1D partial agonists. These receptors are hyperactive, so only slight stimulation is required for major inhibition of pain transmission. Causes vasoconstriction and inhibits trigeminal nerve transmission. 3. Triptans are 5HT1B/D/F agonists. They inhibit trigeminal nerve transmission both peripherally in the ganglia, and centrally in the trigeminal nucleus caudalis. They also inhibit release of vasoactive peptides from meningeal blood vessels. They induce vasoconstriction through 5HT1B receptor activation.

19. Describe neurochemistry and genetic basis of Alzheimer's

1. Acetylcholine is reduced due to decreased synthesis, degradation and uptake, resulting in loss of cells in nucleus basalis which use ACh. 2. Noradrenaline synthesis is decreased due to loss of cells in locus coeruleus and serotonin synthesis is decreased due to loss of cells in raphe nuclei. 3. Possibly reduced glutamate transporters leading to excitotoxicity and potential cause for loss of cells. Amyloid precursor protein (APP) is on chromosome 21 and is the precursor for Abeta. Presenilins (PS1, PS2) on chromosomes 1 and 14 may affect processing of APP and form more toxic Abeta than normal Abeta. Apolipoprotein E (apoE) of e4 and e2 alleles determines the age of onset. 95% of AD has no genetic basis though.

11. What are the different layers of validity criteria of an animal model?

1. Face validity: response observed in the animal model should resemble the behavioural response observed in humans 2. Predictive validity: the animal model should be sensitive to clinically effective pharmacological agents 3. Construct validity: how well theoretical and empirical accounts of the human disorder and the disordered behaviour exhibited by the model are brought into alignment (e.g. behavioural, neurobiological, genetic and molecular features of the human disease)

22. What are the classifications of chronic pain? What other effects are induced by chronic pain?

1. Inflammatory: tissue injury and inflammation processes, often with ongoing nociceptor activity (e.g. arthritis). 2. Neuropathic: lesion or disease affecting the nervous system (e.g. peripheral nerve injury by accident, central injury by stroke or spinal cord damage, diabetes or HIV, and chemothrapy-induced). Spontaneous pain without stimulation, allodynia (normally non-noxious stimuli become painful) and hyperalgesia (reduced threshold to pain response). Results in disturbances in relationships, sleep disruptions, metabolic and endocrine disturbances, reduced movement, loss of interest in external events and depression. Anxiety surrounding this results in additional disturbed sleep and together they cause worse pain.

24. How is opioid addiction treated?

1. Medically assisted detox: clonidine and/or benzodiazepine while providing support to reduce symptoms (high relapse rate though) 2. mu-agonist (metahodone): stops withdrawal and craving without intoxication, because users don't like it as much as heroin. Best with counselling and social support. Some overdose risk. 3. mu-antagonist (naltrexone): occupies receptor and prevents agonists binding, but poor compliance 4. mu-partial agonist (buprenorphine): occupies receptors and prevents full agonists like heroin from binding. Partial agonists reduces craving, also prevents withdrawal. Less overdose risk than methadone. 5. Cognitive behavioural therapy

18. Mechanism of action of antiepileptic drugs (excitation and inhibition)

1. Phenytoin, carbamazepine and lamotrigine enhance sodium channel inactivation, reducing neuron firing frequency: push the channels into conformation that don't allow them to reopen with threshold depolarisation. Specifically target high-frequency firing neurons because channels need to open first and only then can they be pushed into inactivated form (rather than deactivated). 2. Ethosuximide blocks T-type calcium channels to treat generalised absence seizures. 3. Levetiracetam binds to neuronal synaptic vesicle glycoprotein 2A protein, preventing vesicles from coming to the synapse and releasing excitatory amino acids. 4. Barbiturates act on GABAa receptors, increasing affinity for GABA, increasing chloride conductance and prolonging open time of the channel, but a lot of side effects due to non-selective GABAa receptor binding. 5. Benzodiazepines: enhances GABA action by increasing frequency of channel opening. Binds selectively to GABAa receptors with alpha1-3, alpha5 and gamma subunits at the interface between alpha and gamma subunits. 6. Vigabatrin: analogue of GABA, inhibiting GABA transaminase and thus slowing down breakdown of GABA. Tiagabine inhibits GAT1 increasing EC GABA, leading to more GABAa activation. 7. Sodium valproate: sodium channel blocker, calcium channel blocker and increases levels of GABA (unknown how). Topiramate inhibits voltage-dependent sodium channels, antagonises AMPA receptors (reduce excitation) and increase GABA at some GABAa receptors. Felbamate inhibit voltage-gated sodium channels, antagonises NMDA receptors and positively modulates GABAa receptors.

23. Describe the role of 5-HT in migraine

5HT1 receptors are inhibitory, while 5HT2 receptors are excitatory. Patients suffering from migraines have shown to have rapid excretion of 5HT and display disturbances in 5HT metabolism and transmission. 5HT1B can cause vasoconstriction while 5HT2 can lead to indirect vasodilation. The trigeminal ganglion and nucleus have a lot of the inhibitory 5HT1 receptors. 5HT acts on vascular endothelium through 5HT2 receptor, causing release of NO, leading to dilation of extracerebral vessels and sensitisation of sensory nerve endings. These increase sensory nerve discharge, leading to direct pain, central pain sensitisation and neuropeptide release resulting in neuroinflammation.

18. Define epilepsy and describe the different types of seizures

A disorder of the brain characterised by an enduring predisposition to generate epileptic seizures, with occurrence of at least one unprovoked seizure. Focal (partial): neurons activated in a confined region, symptoms reflect the region affected. Complex partial seizures appear as impairment of consciousness (deja vu or strangeness, ticks, hallucinations). Generalised: both hemispheres involved with widespread neuronal activation. Tonic = rigid stretching, atonic = falling to the floor due to loss of muscle tone, clonic = muscle twitching, tonic-clonic = tonic phase followed by a clonic phase (body spasms with intermittent relaxation) and generalised absence seizures = brief lapse of consciousness, people just seem to drift off for a bit.

23. Discern acute from prophylactic treatments of migraine and give examples of drugs used for acute and prophylactic treatment

Acute is to treat occurring symptoms and the drugs are taken after symptoms occur, while prophylactics aim to prevent symptoms from occurring at all, and are meant to be taken all the time. Prophylactic examples: 1. beta-adrenoceptor antagonists - not sure how they work 2. 5-HT2 receptor antagonists prevent 5HT2 receptor-induced vasodilation and consequent inflammation. 3. Anticonvulsants reduce cortical excitability by increasing inhibitory transmission (e.g. GABA) = decrease trigeminal neurotransmission and cortical spreading depression 4. Calcium channel antagonists: decrease calcium entry and thus reduce cellular excitability, decreasing neuropeptide release 5. Tricyclic antidepressants: inhibit 5HT reuptake transporters, increasing 5HT1 receptor activation and thus activating descending pain pathway and reducing activation of ascending pain pathway. 6. CGRP receptor antibodies and peptide antibodies: selectively bind to receptor and protein respectively, reduce ability of CGRP to amplify signals, reducing activation of trigeminal pain pathway.

24. Explain the distinction between drug use and addiction

Addiction only occurs when you're in a state where a drug or a certain behaviour (e.g. gambling) continues in spite of serious potential or actual harm to the user or others. Drug use does not always equal addiction.

17. How is L-Dopa used to treat PD?

Administering peripheral L-Dopa allows to generate dopamine in CNS. L-Dopa is usually metabolised by dopa decarboxylase. Carbidopa or benserazide are administered to inhibit this enzyme in periphery allowing L-Dopa to pass across BBB via a cation transporter, and then the enzyme uninhibited in the CNS can still metabolise it into dopamine. C and B don't cross BBB due to their polarity, ensuring they only inhibit the enzyme peripherally.

14. What treatments are available for multiple sclerosis?

Aim to reduce severity and frequency of symptoms. - Beta interferons: produced in mammalian cells, naturally occurring cytokines. Inhibit viral replication, may restrict lymphocytes crossing the BBB - reduced inflammatory lesions by 50-80%. Side effects: liber, leukopenia, thyroid disease, depression - Sphingosine-1-phosphate receptor modulators (fingolimod): metabolised by sphingosine kinase to active fingolimod phosphate, changing receptor conformation to inactive, causing internalisation of the receptor and preventing migration of lymphocytes from lymph nodes. Side effects: headache, elevated liver enzymes, flu, diarrhoea, back pain - Myelin basic protein mimics (glatiramer acetate): bind up the antibodies that are being directed against myelin and thus prevent the attack from happening, reducing plaque formation. Glatiramer acetate-specific suppressor T cells are induced, reducing inflammation. - Potassium channel blockers (dalfampridine): Na/K exchange is impaired in patients, so by blocking the K channel, the membrane becomes more excited and the effectiveness of sodium channels to maintain APs is increased. Improves walking in MS patients. Overdose causes seizures. - Chemotherapeutic agents (mitoxanthrone): suppresses T cells, B cells and macrophages, impairs antigen presentation and secretion of interferon gamma, TNF-alpha and IL-2. Also potent inhibitor of topoisomerase II which is responsible for repairing damaged DNA (leading to side effects). - Humanised immunoglobulins (natalizumab): IgG4 antibody, binds to alpha 4-subunit of alpha 4beta1 and alpha 4beta7 integrins on surface of T cells and inhibits alpha 4-mediated T cell adhesion. Prevent lymphocyte migration across BBB. - Immunosuppressants: azathioprine and methotrexate disrupt purine biosynthesis, cyclophosphamide given with beta interferon and is a cytotoxic alkylating agent of DNA. Mycophenolate mofetil and cladribine inhibit T and B cell purine synthesis.

6. Understand how the structure of VGICs determine their function

All have 6 transmembrane domains that come in fours, with subunits on each side. The alpha subunits are similar structurally and can function on their own, as they have all the necessary parts. The accessory beta subunits are varied and modulate the function of the alpha subunit's expression levels, location and trafficking (moving to the surface of the neuron). They also alter voltage dependence of activation or inactivation and can bind drugs that modulate function. Additionally, phosphorylating the beta subunit can regulate the channel's function. The aqueous pore of the channel is the selectivity filter (K, Na or Ca). The aqueous channel at the bottom (more IC) facilitates movement of ions our of the channel. The channels contain a voltage centre which moves in response to change in membrane potential - S4 transmembrane domain begins to tilt when the charges in the domain become unstable with positive charges shifting up, opening the bottom outwards and pulling the channel open.

21. Describe endogenous opioids

All have the same N-terminal structure which is the message and then the remaining "address" has variable sequences and lengths and determines the subtype of opioid receptor that it targets. 1. Pre-proopiomelanocortin: gives rise to beta-endorphin and ACTH 2. Preproenkephalin: gives rise to met- and leu-enkephalin 3. Dynorphins: arise from pre-prodynorphin

18. Describe functional effect of a mutation that causes epilepsy

All known mutations relating to epilepsy are in ion channel subunits. Some include glucose transporters, showing that metabolism plays a role - going on diets sometimes helps drug resistance. GABAa receptor mutation: Ly289 switched to methionine in gamma2 subunit of GABAa receptor, decreasing GABAa receptor amplitude, so GABA can still bind but the effects are much smaller. Sodium channel mutation: Nav1.1 Arg1916 to glycine, deems the channel non-functional probably due to protein misfolding. Despite being a sodium channel (which is usually the inhibitory target of epilepsy medicine), Nav1.1 is the sodium channel on GABA neurons so GABAergic transmission stops upon this channel misfolding.

8. How is immunohistochemistry used in neuropharmacology?

Allows NP to look at which receptors/enzymes/transporters/lipids etc. are located the brain region of interest or which receptors may be good targets for disease treatment. Can test whether a drug can change expression of x, change the sub-cellular location of receptors, or change levels of a cellular event such as apoptosis. Tells us about location of the target. Tissue is fixed with formaldehyde and antigen-retrieval is performed to ensure FD did not mask it. Then binding is made specific by using serum albumin to mask unspecific receptors. Detergents are applied to the membrane to make holes so that the antibody can enter the cell and bind to target antigen. To produce desired images, a secondary antibody with a fluorophore attached is used to bind to primary antibody; this binding produces a signal that can be amplified.

14. How does multiple sclerosis compare with other neuroinflammatory conditions?

Alzheimer's: elevated T cells, disrupted BBB, chronic inflammation, elevated Igs associated with vessels in brain tissues and their labelling correlated with neurodegenerative and apoptotic features. Loss of neurons may be due to neuron-specific antibodies (more compromised BBB = more neuron-specific Igs and T cells = cell death). Rheumatoid arthritis: chronic inflammation of joints, treated by anti-TNF (has also shown some effect in Alzheimer's patients) Chronic fatigue syndrome: malaise after exertion, unrefreshing sleep, muscle and joint pain, sore throat, headaches, cognitive difficulties, chronic and severe mental and physical exhaustion. Small pilot study observed treatment with rituximab (mAb against CD20 on B cells, prevent markers on immune cells from working = improved symptoms).

16. Describe antipsychotic drug action and the pathophysiology of schizophrenia relating to the dopamine hypothesis.

Antagonise D2 receptors, with clinical efficacy achieved when receptor occupancy reaches 80% (60-80 for typicals, 40-60 for atypicals). Targeting ventral tegmental area helps reward, social behaviour, salience. Targeting substantia nigra helps movement. 1. Excess levels of dopamine increase neurological activity. Dopamine-releasing drugs resemble positive symptoms and antipsychotic drugs antagonise DA receptors. But no evidence for increased DA turnover and inconsistent changes in post-mortem concentrations. 2. Increased dopamine receptor number or sensitivity. SC brain has more DA receptors and they have higher binding and affinity of DA. However, antipsychotics upregulate DA receptors so this is contradictory. 3. Elevated presynaptic striatal dopaminergic function is the final common pathway in psychosis. There is increased presynaptic DA at nucleus accumbens, and the mesolimbic dopamine system is important in bringing out salience (how important something seems). Over-function can lead to increased salience of stimuli - normal stimuli appear frightening. 4. GABAergic deficit in hippocampus underlies DA dysregulation. Post mortem brains have shown less GABA neurons = unrestrained excitability especially in ventral hippocampus, which feeds into the medial prefrontal cortex and nucleus accumbens. More hippocampal activity = more dopamine release.

11. How are the animal models used to study anxiety disorders?

Anxiety measured through observing the animal's tendency to explore a new environment and to avoid an open area. - Open field test: measure the amount of time mice spend in the perimeter compared to the centre of the box (centre = less anxiety) - Light Dark (or Emergence) test: will spend more time in the light in the open space if low anxiety (high anxiety will be in a dark box portion) - Elevated plus maze test: low anxiety would result in an increase of time spent in open arms of the maze compared to arms with walls - Social interaction: will interact more at lower anxiety levels - Innate fear through predatory odour avoidance: exposed to cat odour during conditions leading to innate response of anxiety. Treatment of midazolam during conditioning reversed this anxiety, as seen by reduced time spent hiding.

2. Describe the roles of the different types of glial cells

Astrocytes make up 25-50% of cell volume in most regions of the brain. Filaments provide structure to the brain, extending processes to the cell bodies of neurones. Also form part of BBB. Help maintain neuronal haemostasis (ion gradients, NA/K concentrations, etc.), secrete neutrophic factors (specific direction, allow neurones to connect correctly), regulate EC environment through transporters, and store glycogen (energy). Also regulate neuronal migration and axon and dendrite growth. Oligodendrocytes and Schwann cells produce myelin, providing insulation around the axon for rapid conduction of signals Microglia are immune cells of the brain and spinal cord, defending against infection. Change morphology and number during insult or injury, resulting in an inflammatory response.

19. Describe clinical features and pathology of Alzheimer's

At first just slight changes in memory and cognition like forgetting words or losing things. Then spatial and temporal orientation changes and difficulty forming new memories, altered judgement. Increased moodiness, depression and apathy. Eventually impaired speech, movement, loss of knowledge and often paranoia and psychosis. Pathology: cerebral atrophy and ventricular enlargement due to overall massive shrinking of brain tissue. Neuronal loss with glial proliferation forming scars in hippocampus, cortex, amygdala, olfactory system and nucleus basalis. Amyloid plaques from EC amyloid protein (mainly amyloid beta) aggregates in neocortex and hippocampus. Neurofibrillary tangles (abnormal bunches of filaments in neurons and hyperphosphorylated tau protein).

2. What is the blood brain barrier, how does it regulate the brain environment, and how does it influence drug actions in the brain?

Barrier made of endothelial cells with tight junctions in between, separating the brain's EC fluid and general circulation. It's a continuous lipid bilayer, of capillaries in cerebral vascular beds. Helps control conditions precisely and prevents entry of potentially toxic substances. Drugs need to either be prodrugs (group attached for lipophilic entry and then cleaved to active drug inside the brain) or lipophilic drugs so they can cross the BBB membrane.

20. Describe and explain the mechanism of action of benzodiazepines at the synaptic and regional levels

Bind in between the alpha and gamma subunits in the GABAa receptor, specifically selective for receptors containing alpha1, 2, 3 and 5. It increases the affinity for GABA and thus increases the frequency of chloride channel openings. Results in higher inhibition of neurons. Same amount of GABA release will cause a much greater inhibitory effect post-synaptically. alpha1 selection is sedative/hypnotic, while alpha2 and 3 are anxiolytic and alpha3 and 5 relax muscles. BZ use reduces total REM stage leading to hypnotic and sedative effects. Also anxiolytic and anti-epileptic.

15. MOA of tetracyclics (e.g. amitriptyline and imipramine)

Block alpha2 adrenoceptors presynaptically, leading to increase NA and 5-HT release. 5-HT2 and 5-HT3 are blocked postsynaptically, so preferred action on 5-HT1.

5. Describe the role of transporters from the glutamate transporter family and the NSS family and the drugs that interact with these transporters

Both are secondary active transporters. Glutamate transporter family: clear glutamate/aspartate from the synaptic cleft. EAAT2 is the main one that pumps glutamate out of the synapse into the neighbouring astrocyte cell. Couple glutamate with sodium and hydrogen electrochemical gradient, helping maintain 10^6-fold gradient across the membrane. L-TBOA is a competitive inhibitor, binding to the site and preventing the lop from closing down. The closing is required for the process to continue, so this drug acts as a blocker. Neurotransmitter Sodium Symporter (NSS) family: clear GABA, glycine, dopamine, noradrenaline and serotonin from the synaptic cleft. - Glycine is coupled to sodium and chloride co-transport - GlyT1 inhibitors (e.g. sarcosine) can elevate glycine at excitatory synapse and help stimulate NMDAR in schizophrenia patients. GlyT2 inhibitors increase glycinergic inhibition in the spinal cord, reducing excitatory pain signal transmission (e.g. ALX1393). - GABA: tiagabine selective for GAT1 as an anticonvulsant (inhibit transporters = elevate GABA = less sedation) NSS also targets for drugs of abuse: amphetamine enters the cell in exchange for dopamine, resulting in elevated D in the synapse and elevated A in the cell. Cocaine blocks dopamine, NA and Ser reuptake.

4. Explain why corticotrophin releasing factor (CRF) might or might not be a good target for psychotherapy

CRF is vital in observing behavioural and metabolic responses to stress, seen as stress favours depression and anxiety, and CRFR1 mutations have been shown to be associated with depression and anxiety also. CRF administration in mice has shown to increase behavioural stress, so they were hoping that CRFR1 antagonists would suppress behavioural changes associated with stress. While this worked for mice, it did not consistently show significant results in studies with humans. This is possibly due to a very narrow population of subjects used, poor animal models (forced swim test to see how depressed the rodent is = human experience of depression is very different to a rodent), and there are no pet ligands for CRFR1 to allow determination of drug concentration that should be used.

16. Recognise the principle drugs used in the treatment of schizophrenia. Outline their pharmacological action, clinical efficacy and side effect profiles.

Chlorpromazine and thioridazine antagonise D2 receptors, highly sedative. Also haloperidol. By blocking D2 autoreceptors, this increases DA release; blocking supersensitive postsynaptic D2 receptors decreases DA inhibition. Alleviate positive symptoms but little effect on negative and cognitive symptoms. Also have moderate affinity for adrenergic and 5-HT2 receptors and weak affinity for D1, D4, H1 and mACh receptors. Clozapine and olanzapine have high affinity for D4 and clozapine dissociates rapidly from receptors (perhaps why it's effective in treatment-resistant patients, due to less tolerance developed).

19. What are the treatment options for Alzheimer's? Give examples, both current and experimental.

Current 1. Cholinesterase inhibitors: some reversible, some non-competitive, some allosteric, etc. However can treat symptoms but not halt progress of disease because need ACh neurons to release ACh in the first place. If neurons are already lost, these drugs are ineffective. 2. NMDA antagonists to treat glutamate toxicity 3. Antidepressants and neuroleptics modulate NA and serotonin transmission and help anxiety and depression, but don't affect core symptoms. Experimental 1. beta-secretase inhibitors, preventing amino acids to be cleaved so Abeta42 (toxic amyloid protein) cannot be made effectively. gamma-secretase inhibitors bind to asp-asp catalytic site, preventing activity. They both prevent or disrupt formation of Abeta plaques. Gamma are easier to make but have more side effects due to cleaving many other proteins. 2. Statins to reduce cholesterol synthesis because elevated levels may disrupt processing of APP, leading to outweighing APOE4 production, decreasing age of onset. 3. Chelating zinc reduces plaque formation (animal studies) 4. Immunisation with Abeta so the immune system recognises it as foreign and prepares an immune response for the future (only seen in mice, so far toxic in humans)

17. What are PD drug treatments alternative to L-Dopa?

DA agonists (bromocriptine, pergolide, pramipexole and rapinirole): stimulate D2 in striatum Monoamine Oxidase B (MAOB) inhibitors (selective selegeline): reduce L-Dopa and dopamine metabolism, increasing L-Dopa's bioavailability so a smaller dose is needed. Muscarinic antagonists (benztropine, trihexyphenidy, diphenhydramine): reduce GABA output, helping with tremor and rigidity, can be used to treat drug-induced dyskinesia but not as effective against PD as L-Dopa. NMDA receptor antagonists (amantadine): promotes dopamine release but unclear how. No significant side effects but smaller effect and for less time.

3. Give examples of drugs that target monoamine transmission, explain their MOA and their therapeutic use.

DA: D1-D2 agonist apomorphine for Parkinson's disease A: clonidine (partial agonist for BP), mirtazepine (a2 antagonists for depression) 5HT: busripane (1A agonist for anxiety), clozapine (2A antagonist for psychosis treatment), ondansetron (3 anatgonist for antiemesis), cisapride (4 agonist for gastroesophageal reflux)

6. Identify mechanisms for control of VGICs

Deactivation: membrane potential reverses to resting state a couple of seconds after opening = channel closes. Inactivation: channels close immediately after being activated (even in the presence of depolarisation). Can only become reactivated when the IC region dissociates from the core of the channel. Phosphorylation: allows for rapid changes in channel function (seconds or less) by altering the channel so it responds to different physiological stimuli. Protein kinase C phosphorylates one residue in alpha subunit of a sodium channel to slow the rate of activation, thus keeping the channel more likely to be subsequently activated by a voltage signal. Protein kinase A can slow the rate of calcium channel activation and shift its voltage dependence so that it's activated at more negative potentials.

2. Identify the key cellular features of a neurone. Include the locations of VGIC, LGIC, GPCR, transporters, myelin sheath, dendrites, axon, soma, etc.

Dendrites: long processes, varying degrees of complexity, carry signals to the cell body (soma), where they combine at the axon hillock to produce one single signal. If degree of depolarisation is sufficient, this generates an AP. Axons is coated in myelin sheath with nodes of Ranvier in between with series of sodium and potassium channels (VGIC) to renew AP again and again to keep the signal traveling through. Signal reaches axon terminal/bouton, vesicles fuse with post-synaptic membrane to release neurotransmitters to activate GPCRs in the postsynaptic density (receptors directly opposite to ensure undistorted clean signal transduction). Synapses can be axo-dendritic or axo-somatic, axo-axonic, autoreceptors (self), or involve multiple neurotransmitters in a complex system of synapses.

4. Define how neuropeptide neurotransmitters/modulators differ from other types of neurotransmitter systems (e.g. glutamate)

Different vesicles (DCV = dense core vesicles), much larger and appear to be darker due to the density. Release is not as sensitively stimulated by calcium because DCVs are released from terminals or dendrites, which are far away from calcium. Instead, receptors have really high affinity, allowing these messages to travel much further across the brain (while glutamate and GABA have to be close to their receptors). There are many more points of modulation - one prepropeptide can result in many different neuropeptides by the time they are ready to be released from the vesicles. Messages are modulated and they are much slower compared to glutamate/GABA release. There is no reuptake because of this, because it isn't as important to get rid of the peptide quickly due to slow signalling. Instead only degraded by EC peptidases.

11. How are the animal models used to study Dravet Syndrome (childhood epilepsy)?

Disease: impaired SCN1A due to a mutation - voltage-gated sodium channel that regulates GABA release; can introduce mutation in form of KO mouse. A febrile event (fever) can cause the first seizure and then progress to spontaneous seizures and eventually death. Animal model testing: heating them up a bit to see if a seizure will be caused, spontaneous seizure observation, measuring resulting lifespan and behavioural abnormalities (hyperactivity, impaired memory function, social recognition deficits).

24. Describe some of the cellular adaptations that produce addiction

Drug use causes extremely high activation of dopamine release, thus causing a massive experience of reward on a different scale from natural or endogenous reward. Changes in amygdala and hippocampus affects learning; they are tightly linked to nucleus accumbens and the ventral pathway and together this results in a learnt association between drug and pleasure. Changes in orbitofrontal cortex (motivation and drive) and prefrontal cortex and anterior cingulate cortex (cognitive control) leads to poor decision making and continuous drug use. When a drug causes an effect, the brain works to induce tolerance by developing adaptations to try and get the balance back to normal. This results in higher drug use to experience the same effect, and more adaptations occur to balance it again. Upon withdrawal, the adaptations outweigh the drug effect, e.g. in heroin use, ncreased GABA release to balance out dopamine = upon withdrawal from heroin it is a lot more inhibition causing major dysphoria. Adaptations include increased adenylate cyclase activity and inhibition of phosphodiasterase upon morphine use. Also there can be large changes in neurotransmitter release through major increase in adenosine.

8. How is electrophysiology used in neuropharmacology?

EP measures changes in membrane potential, ultimately measuring movement of ions across the membrane (in/out of the cell). As an AP propagates into the cell, it causes ions to go in and out of the cell, resulting in voltage changes which can be measured by inserting reference electrodes that are connected to an amplifier (which displays the changes). However, difficult to exchange drugs or to have good electrical control of the cell in neurons. Developed patch clamping. In NP, can help observe how drugs modify ion channel action, change neuronal excitability, change synaptic transmission and synaptic plasticity. Can also show where the drugs are acting and how drugs interact with mutated proteins.

11. How are the animal models used to study unipolar depressive disorder?

Forced swim test or tail suspension test can be used to see how long it takes the mouse to stop swimming and give up, representing behavioural despair - time decreased in depressed mice. Immobility reduced with desipramine treatment and P2X7 KO model (latter may be a good target). Learned helplessness through chronic unpredictable stress. Also look at chronic mild stress and novelty-induced hypophagia (reduction in food intake and eating behaviour)

7. Explain the action of some therapeutic drugs on LGICs are the molecular, cellular and behavioural levels

GABAa: benzodiazepines enhance the actions of GABA by binding to GABAa receptors, making the channel open and close more frequently by binding at the interface between alpha and gamma subunits. Sedation = alpha1, anxiolysis = alpha2, muscle relaxation = alpha2 and 3. Barbituates make the channel open for a longer extended period of time to enhance GABA action. Glycine: ivermectin binds within the membrane and picrotoxin binds specifically within the pore of the channel (selective for homomeric receptors, as beta subunits have different amino acid substitutions compared to alpha, changing the environment of the channel = picrotoxin can no longer interact). 5HT3: alosetron (irritable bowel syndrome) and ondansetron (helps against nausea in chemotherapy and often in pregnancy). Bind as competitive antagonists between alpha and beta subunits. Not good to use glutamate agonists because will lead to over-excitotoxicity and cause seizures. Antagonists can be used to help in pain relief (e.g. ketamine and phencyclidine) but have many side effects such as hallucinations.

5. Describe the structure of the homologues of both families

Glutamate family homologue is from Pyrococcus horikoshii. It is a trimer (3 identical subunits), each unit capable of transport. Has a deep bowl-shape structure. Instead of glutamate, model it with aspartate. NSS: homologue of dopamine transport (Drosophila DT). Has 12 transmembrane domains in a shot glass shape, the substrate buried in the middle. Co-transports 2Na and 1Cl.

21. What are the main opioid drugs and how do they differ?

Heroin is a pro-drug and has acetyl groups instead of hydroxyl groups attached. This allows it to cross the BBB very rapidly in inactive form, where it can then be metabolised by esterases to active form into morphine. Codeine is nearly completely inactive with methyl group instead of hydroxyl, but once this is hydrolysed by CYP2D6, it turns into morphine which accumulates in higher amounts. Buprenorphine is a partial agonist, so it binds very tightly to the receptor but only produces a weak G-protein signal. The tight binding still prevents other agonists/antagonists from binding. Useful in tolerance because will produce zero effect so can almost act as an antagonist - can't kill addicts because receptors can't be stimulated enough.

14. What is multiple sclerosis?

Inflammatory disease where myelin sheath is damaged around the axons, impairing nerve conduction.

15. MOA of monoamine oxidase inhibitors (e.g. moclobemide)

Inhibit breakdown of NA, 5-HT and DA by inhibiting enzymes, thus increasing cytoplasmic pool of monoamines. This increases leakage of monoamines into the synapse and EC space.

17. Mechanism of action of drug-induced PD

MPPP (sold as synthetic opioid) containing impurity MPTP, which crosses BBB and is converted to MPP+ by MAOB. MPP+ is then taken by dopamine transporter, inhibiting oxidative phosphorylation and tyrosine hydroxylase, affecting mitochondria function. Produces same effects as PD like jerking of limbs and stiffness, with difficulty moving and after 6 weeks near total immobility, inability to speak, and tremor at rest. Post-mortem showed loss of neurons in substantia nigra and presence of Lewy bodies, giving an insight into Parkinson's pathology.

20. Outline the problems with the use of benzodiazepines

Maximum length of use should only be 1 month but it's still unclear if longer use is problematic. Upon stopping, use should be lowered gradually and dependence liability should be considered. Use can cause strange sleeping behaviours, amnesia, falls in elderly and combination with other sedatives can be dangerous.

15. What is the cause of depression? What is the link between depression and stress?

Monoamine concentration at synaptic sites are significantly lower, so blocking re-uptake is a direct target for antidepressants to increase this concentration. Effects take 3-4 weeks to be experienced by the patient. Gut microbiota has been shown to also potentially play a role - giving faecal transplants from depressed patients to mice resulted in behavioural changes in depressive symptoms. Chronic stress in depression inhibits negative feedback and results in high cortisol in blood due to constant release. Cortisol receptors become desensitised, increase inflammatory mediators and disrupting neurotransmission. Can lead to adrenal hypertrophy and hippocampal atrophy. Loss of synaptic connectivity has been shown through reduction in contacts points, which can affect cognition, etc.

16. What is the potential role of glutamate in schizophrenia?

NMDA receptor antagonist PCP has shown to cause both positive and negative symptoms and NMDA receptor KO mice show schizophrenia symptoms. Show changes in glutamate receptor and transporter expression in prefrontal cortex and temporal lobes, with reduced pyramidal cell dendrites and dendritic spines. Seen with low glutamate concentration. Can enhance glutamatergic activity through glycine transporter blockers and partial agonists. Cannabidiol (CBD) showed efficacy in reducing positive symptoms without side effects - can boost endocannabinoid production in the brain. High anandamide concentration in CSF shows that the body is trying to upregulate production to fight disease - possible future target.

2. Describe the role of the Na/K-ATPase and the KCC2 co-transporter in setting the resting potential of a neuron.

Na/K-ATPase uses energy from ATP hydrolysis to bring potassium in and sodium out of the cell. It also helps drive chloride out together with potassium through the KCC2 co-transporter, as potassium begins to travel with the concentration gradient to balance out the Na/K-ATPase. Large impermeant anions also always stay in the cell, thus responsible for the resting potential by balancing out this movement of ions.

22. What is the current treatment of chronic pain?

Opioids: don't work as well because mu receptors are only on nociceptive neurons, not all sensory neurons and in chronic pain non-nociceptive neurons become noxious. However, still act on mu receptors in ascending pain pathway so some relief provided. Tricyclic antidepressants: they block the re-uptake transporters. Serotonin and NA in spinal cord cause inhibition of the ascending pathway, so SSNRIs (dual NA and 5HT) help pain relief. 5HT activates central descending pathway and NA inhibits ascending pathway through alpha-2 receptor activation (reduces glutamate release from nociceptive nerves and directly inhibits pain pathway on dorsal horn neurons) VGCC channel blockers: wide distribution throughout afferent nerves, so better pain relief but also side-effects. Delivered intrathecally (spine) to reduce these. Calcium channels are overexpressed in chronic pain, so by blocking them calcium does not leave and presynaptic neuron does not get depolarised so neurotransmitter does not get released. GABA analogues (gabapentin, pregabalin): cross BBB due to being similar to GABA, so can get into brain and spinal cord. However, does not bind to GABA receptors of the BZ binding site due to being less flexible. Binds to alpha2delta-subunit in VGCCs, preventing trafficking of VGCC to membrane and reducing channel activation. Slower process compared to blockers or agonists. Tramadol: partial opioid agonist and SSNRI

5. Understand the difference between passive and active transport and give examples of each

Passive: requires no energy, moves along the electrochemical gradient, e.g. AMPA glutamate receptor. Active: transporters can move substrates against a huge electrochemical gradient, and this requires energy. When they get their energy from ATP hydrolysis, this is primary active transport. Can use ATP pumps, such as Na/K ATPase. Coupling to other ion gradients is secondary active transport, e.g. plasma membrane glutamate transporter couples glutamate with sodium in exchange for potassium.

7. Give examples of LGICs and their endogenous ligands.

Pentameric: nACh excitatory, 5-HT3 excitatory, GABAa inhibitory and glycine inhibitory receptors. Ligands include acetylcholine, serotonin, GABA and glycine. Glutamate: AMPA, kainate and NMDA excitatory receptors. Ligands include AMPA, kainate, NMDA and glutamate.

7. Explain how changes in LGIC structure can alter function

Pentameric: the M2 pore lining helices contain charged residues. When the helices bend towards the centre of the pore, this creates a hydrophobic pocket that doesn't allow water molecules in and narrows the channel, essentially closing it. When the helices rotate or tilt, this opens the pore and thus the channel. Ionotropic Glutamate: as glutamate binds to LBD, this snaps the region shut, shifting the gate apart, allowing channel opening.

7. Identify the two main classes of LGICs, describe their structure and explain how these channels are gated.

Pentameric: usually have 2 ligand-binding sites. When ligands bind, this opens the channel. Consist of 4 transmembrane domains, one of them being intracellular (2). Domain 2 forms the lining of the tunnel through which the ions pass. Ionotropic Glutamate: consist of the amino terminal domain, ligand-binding domain and transmembrane domain. AMPA receptors only require glutamate to bind to be activated, while NMDA receptors need glutamate and glycine to be activated. AMPA are fast and selective for sodium. NMDA are slow, allow sodium and calcium through, and have voltage-dependent magnesium block. Sodium influx through AMPA results in depolarisation of the membrane, freeing the voltage-dependent block by release magnesium into the EC solution. This now allows ions to flow through the open channel.

11. How are the animal models used to study schizophrenia?

Positive symptoms (hallucinations, delusions and bizarre behaviour) are modelled through dopamine agonist treatment (because DA increase has been shown in schizophrenia) and NMDA antagonist treatment (show hyperactivity and stereotypy, run around a lot more). Negative (apathy, anhedonia, social withdrawal): models of anxiety, reduced sucrose consumption, observing social interaction Cognitive (impaired attention and memory): prepulse inhibition of startle (pre-exposure to a weak stimulus reduces the startle response to a loud stimulus = SC patients show poor PPI) and Y maze and Morris water maze.

23. Describe the role of trigeminovascular system in the generation of migraine

Possible cause is a transient, intense wave of excitation due to high K+ leading to a cortical spreading depression/profound inhibition. The spread activates the trigeminal nucleus caudalis, causing sensitisation of central pain pathways. Trigeminal ganglia send their sensory fibres to the meninges and the major blood vessels surrounding the brain, resulting in widespread pain. Activation of trigeminal nerve fibres is also possibly due to vasoconstriction. Cortical excitation causes pain directly and release of CGRP (and substance P, NO, NKA) causes vasodilation and dura mater neurogenic inflammation (causing protein leakage, mast cell activation, prostaglandin release, etc.). This leads to increased activation of the ascending trigeminal pain pathway.

4. Describe the basics of endogenous opioid neurotransmitter system

Responsible for regulation of mood, fear response, pain perception, GIT function, decision making, drug addiction, attachment formation and stress response. Have shown to inhibit glutamate release and thus reduce level of post-synaptic excitation. Agonist action has shown to result in pain relief and euphoria but also constipation and resp. depression. Antagonists or knock out mice have shown to increase stress, showing high anxiety and fear behaviours. While stimulating the brain in the central/periaqueductal gray area produced analgesia, naloxone reversed this - shows presence of endogenous opioids. POMC originates in hypothalamus but is spread across the whole brain through axon processes; Penk acts locally and so its axon locations match its cell body locations.

20. Dissect sedative-hypnotic from anxiolytic drug classes

Sedative-hypnotic drugs increase major inhibitory neurotransmitter acton leading to sedation. Used to treat insomnia and other sleep disorders. Anxiety occurs due to the brain preparing for danger (arousal, vigilance, increased ability to respond) but without any threatening stimulus, affecting daily function. Amygdala activity is up-regulated, possibly due to changes in synaptic plasticity in amygdala circuits. BZ still used but for more acute situations and not as first line of treatment. Alprazolam and clonazepam have high alpha2 affinity (helps anxiety specifically). Serotonin reuptake inhibitors and agonists can help as well, but take weeks to cause change that can be noticed.

15. MOA of selective serotonin reuptake inhibitors (e.g. fluoxetine, paroxetine, sertraline)

Selective block serotonin transporters, preventing re-uptake and increasing serotonin in the synapse. Don't affect mACh and histamine receptors, so fewer side effects. However, increased serotonin action of 5-HT receptors can still result in side effects.

6. Understand how drugs manipulate the actions of VGICs

Sodium: need to be selective (or results in loss of sensory system and coma). Sodium channels have 9 different types of alpha subunits, differing in their distribution throughout tissues. Tetrodotoxin binds to external surface of S5-S6 loop region, thus blocking the selective pore and blocking the channels. Phenytoin and carbamazepine treat epilepsy by slowing the recovery from the inactivated state (limiting firing rates of neurones and preventing seizures). Local anaesthetics binds to the site exposed to the lipid membrane, thus blocking the channel. Potassium: noradrenaline increases firing activity of hippocampul pyramidal neurones (beta - cAMP production - Protein kinase A) by phosphorylating calcium-activated potassium channels and blocking their activity, leading to hyperpolarisation and keeping the neuron in an excitable state. Tetraethylamonium inhibits most potassium channels (research) and caesium inhibits most as well. Calcium: gabapentin and pregabalin used for treating chronic pain and epilepsy by binding to the alpha2delta subunit and preventing trafficking of the channel to the surface (selective for CaV2.2., which is responsible for neurotransmitter release in sensory neurones).

17. Symptoms, etiology and pathology of PD

Symptoms: tremor and rigidity, bradykinesia (slow movement), postural instability. Also cognitive, mood, autonomic dysfunction, motor fluctuations and late dementia. Due to immobility, death is often because of falls or pneumonia because unable to cough out the infection. Etiology: cause mainly unknown, but is a progressive neurodegenerative disease. Genetic factors thought to play a part in 5-10% cases but not present in sporadic cases (these are more likely at a younger age). Possibly environment: pesticides, heavy metal poisoning, viruses, etc. Pathology: low dopamine due to loss of dopamine neurones (substantia nigra to striatum) and cannot notice symptoms until 80% dopamine reduction. Losing DA neurones removes inhibitory effect from GABA release, increasing GABA output. Lewy bodies are formed, made from oxidised chemicals that either aggregate the pathogenesis or are perhaps breakdown products of it.

3. Explain the normal synaptic synthesis, storage, release, receptor action and inactivation of monoamines.

Synthesis: - Catecholamines: precursor L-tyrosine converted to L-DOPA by tyrosine hydroxylase. L-DOPA converted to dopamine by decarboxylase, dopamine converted to NA by dopamine beta-hydroxylase and NA converted to adrenaline by PNMT. - Serotonin: L-tryptophan to 5-HTP to 5-HT Storage: enter vesicles with active transport via vesicular monoamine transporter, stored as complex bound with ATP. Release: AP depolarises terminal, allowing calcium entry and fusion of vesicles with presynaptic membrane = neurotransmitters released into synapse. Serotonin can be released extra-synaptically and be taken up by astrocytes because its receptors are widely distributed (hence long serotonergic release). Receptor activation: - Dopamine: D1 excitatory and D2 inhibitory - GPCR adrenoceptors: adrenaline mainly on beta2, NA mainly on alpha. - Serotonin: 5HT3 ionotropic (rest metabotropic), 5HT1 mediate inhibition (the rest mediate excitation) Inactivation: - Reuptake through active transport via high affinity sodium-dependent membrane transport proteins for dopamine, NA and serotonin. - Degradation: broken down by monoamine oxidase via oxidative deamination. MAO-A (5-HT, NA, A, DA) and MAO-B (DA).

3. Describe the drug targets of monoamine neurotransmission.

Synthesis: L-DOPA helps in Parkinson's disease to increase dopamine synthesis in the substantia nigra Storage and release: amphetamines compete with monoamines for storage in vesicles, so increased cytoplasmic monoamine increases spontaneous leakage into the synapse. Reuptake: amphetamines (e.g. MDMA) prevent reuptake, leaving the NT in the synapse for longer for prolonged receptor stimulation. Antidepressants such as bupropion blocks NAT and DAT. Degradation: MAO inhibitors used to treat depression, selegiline (MAO-B inhibitor) used to treat Parkinson's by increasing DA in the brain.

22. What are the general mechanisms of analgesia and what changes in chronic pain?

The ascending pathway is from periphery to spinal cord to brain. Activation leads to perceived pain intensity through higher processing. Descending this pathway produces analgesia. The descending pathway is responsible for shutting down the ascending pathway through neurotransmitter release. Injury of peripheral afferent nerves leads to neurotrophin release (growth factor in immune response) and this causes changes in the surviving nerves, including both non-noxious and nociceptive neurons. Changes can cause reduction in threshold and increased sensitivity. Central changes result in nerve sprouting - fibres that weren't in pain circuits before are now going in, resulting in a higher response. There is also increased excitatory transmission and reduced inhibition, and changes in the brain leading to altered brain behaviour.

6. Understand the role of VGICs in synaptic transmission

The voltage-gated sodium channel opens due to depolarisation of membrane potential, then letting through sodium ions into the cell leading to further depolarisation. Depolarisation also opens potassium voltage-gated channels, allowing potassium to flow out of the cell, further speeding up rate of depolarisation. This causes membrane potential to overshoot the resting state and thus reach the threshold needed to generate a signal.

14. What are the pathogenic features and symptoms of multiple sclerosis?

There are multiple scars of plaques or lesions in the CNS, commonly in the myelin sheath of the optic nerve, brainstem, basal ganglia and spinal cord. Episodes at varying time intervals in different parts of the CNS. No single symptom to diagnose. Symptoms affect CNS (fatigue, cognitive impairment, depression, mood), vision, speech, throat, musculoskeletal system (weakness, spasms), sensation (pain), bowel movement (loss of control, diarrhoea or constipation) and urinary system (frequency, loss of control). MRI used for diagnosis, can clearly show plaques. Cause is unknown but possibly there is a viral infection causing BBB to become leaky, allowing immune cells through and thus attacking myelin sheath as foreign (as they have never encountered it before). Inflammatory mediators are released cause inflammation, thus causing build up of plaques. After infection is cleared, BBB is fixed but later becomes leaky again (unclear why), hence why damage is intermittent.

21. How is pain transmitted?

There are specific sensory nerve pathways that get activated by noxious stimuli that go from the spinal cord to the brain. There are A-delta fibres and C-fibres. The A-delta fibres are fast myelinated neurons and produce the first spike of painful experience. The C-fibres are slow unmyelinated neurons and produce a slow second wave of pain.

4. Basic properties of neuropeptides as neurotransmitters/neuromodulators

They are small proteins that act as NTs in the nervous system. Linear polymers made of amino acids joined by peptide bonds.

15. MOA of monoamine reuptake inhibitors (e.g. venlafaxine)

They block presynaptic noradrenaline and/or serotonin transporters and thus increase NA and 5-HT in the synapse. Also block mACh, 5-HT, histamine and alpha-adrenergic receptors (hence many side effects).

5. Understand what membrane transporters do and how they are different to receptors

They can be passive or active (receptors are only passive) They transport substrates across the cell membrane (in receptors, substrates can activate a GPCR and cause a cascade, or they can bind to ligand-gated ion channels to let ions through).

3. Describe the diversity of CNS functions mediated by monoamines.

They help regulate mood, reward system, movement, pain, autonomic function and hormone release. Very important in anxiety, schizophrenia and drug abuse. Extremely diverse because pathways are so variable and spread out across different regions of the brain, all of which are highly specialised in their function. Dopamine: hormonal regulation, movement, cognitive control, motivation, emotional responses, reward pathway NA: pain modulation, movement Serotonin: arousal, mood, appetite, temperature

21. What is the mechanism of action of opioid drugs?

They mainly act on C-fibres because opioid receptors are mainly located here. Opioid receptors are only located on nociceptive neurons so highly selective. Act on the forebrain lateral sensory system, the forebrain medial system emotional response, midbrain and brainstem (descending) and the spinal cord. The alpha subunit of the GPCR dissociates to bind to calcium channels, inhibiting neurotransmitter release. Beta-gamma subunit dissociates to bind to GIRK potassium channels, increasing their activity, thus blocking APs and hyperpolarising the cell membrane.

16. Differentiate atypical from typical antipsychotics in terms of chemical classes, clinical efficacy and side effect profiles.

Typical are phenothiazines (chlorpromazine and thioridazine), butyrophenones (haloperidol) and thioxanthenes. Only help alleviate positive symptoms, require 80% receptor occupancy for clinical efficacy. Side effects in most categories, especially movement, anticholinergic effects and endocrine. Atypical are diazepines (clozapine and olanzaine), dibenzothiazepines (quetiapine), benzamides, benzisoxazols (risperidone) and quinolinone derivatives (aripiprazole). Help alleviate both positive and negative symptoms. Fewer side effects, only require 60% receptor occupancy for clinical efficacy. Side effects mainly in agranulocytosis (reduction in white blood cells) and metabolic syndrome.

11. How are the animal models used to study PTSD?

Use mice to look at anxiety, increased conditioned fear to context (exposure to electric foot-shock; putting mice back in the box increases freezing behaviour), fear potentiated startle and decreased hippocampal volume.

8. Why is patch-clamp electrophysiology is the dominant type of electrophysiology used in neuropharmacology/neuroscience?

Usually a microelectrode is pushed through the cell membrane, creating a hole in the membrane which resulted in a lot of noise and it was hard to control it. Patch-electrodes have a much larger tip, which you can attach to the cell by slightly sucking a bit on the electrode, sucking a bit of the membrane back up the glass and then clamping the membrane so the electrode is attached without making a hole. Allows to control what's inside the cell exactly, as when you break the pipette, whatever was in it goes directly into the cell, as there is no hole. Results in very low noise and high resolution of currents. Can make single-channel recordings, very precise. Can show how drugs alter synaptic transmission (e.g. applying an opioid agonist can show how synaptic transmission of glutamate is reduced in specific receptors. Also allows to test a drug for its efficacy on the learning process. The method is versatile (several proteins measured in the same cell or can look at single cell, cultured cells with mutated proteins, slices, in vivo). Allows access inside of the cell, so can manipulate the signalling systems of one cell at a time. Voltage clamp allows measurement of voltage-dependent ion channel responses. By injecting a neg/pos current through the amplifier and then applying an agonist to open channels (e.g. potassium, this allows for ion efflux and thus the change of membrane potential. To balance this, the amplifier has to apply a higher current to keep the steady state of membrane potential constant = these changes are recorded. Upward deflection represents positive ions leaving, while downward deflection represents negative ions leaving the cell. Current clamp measures the change in membrane potential caused by the applied current (rather than keeping the potential constant and changing the current).