I decided to take Drugs and the Brain, my third Coursera class, late last year when I saw it in the list of offerings. The 7-week course by Henry Lester of the California Institute of Technology began on January 5.
Week 1
Videos 1 and 2 provide an overview of the course.
Video 3, What is a drug, presents information about nicotine, lidocaine, morphine, and botulinum toxin as examples of drugs. In the context of the brain, drugs:
Week 1
Videos 1 and 2 provide an overview of the course.
Video 3, What is a drug, presents information about nicotine, lidocaine, morphine, and botulinum toxin as examples of drugs. In the context of the brain, drugs:
- Activate (e.g., nicotine) and block (lidocaine) ion channels.
- Act on G-protein coupled receptor pathways (e.g., morphine).
- Activate genes (e.g., nicotine, morphine).
- Protein drugs (e.g., botulinum toxin) may become more useful for neuroscience diseases.
- Act on neurotransmitter transporters.
The term moiety was defined as a group of atoms in a drug molecule. Moieties have importance in that charged amines can bind to oppositely charged groups or the pi electrons of receptor proteins, amides can be hydrolyzed to terminate drug action, aromatics can bind to nonpolar groups on receptor proteins, and alkyl substitutents can adjust charge density of amines, affect membrane permeation, and affect receptor binding. Counterions and salts (e.g., HCl) are added to make drugs more soluble.
Video 4, Drug Entry into the Nervous System, discusses routes into the body, such as lungs (smoking), chewing, patches, injection, suppositories, and application in creams. Drugs such as alkaloids enter the body and diffuse through membranes in neutral form (RNH2) but act on receptor in charged form (RNH3+). Ammonium hydroxide is added to some cigarettes to keep nicotine neutral so more permeates to the brain. Parkinson's disease is treated with levodopa, a prodrug (drug that is converted into another drug in the body) that is a zwitterion (contains both positive and negative groups) that is converted by carboxylases into dopamine in the brain. Dopamine cannot cross the blood brain barrier (BBB) but once in the brain is taken up by dopaminergic neurons for use. The BBB consists of capillaries with tight junctions found only in the brain (holes are present in other parts of the body) that zip endothelial cells together so that only certain nonpolar and hydrophobic molecules can pass, via the endothelial cell membranes.
Video 5, Introduction to Drug Receptors...A very basic lecture: A receptor is a molecule on the cell surface or interior with affinity for a drug or ligand (Greek: to tie). Most receptors are proteins. Amide or peptide bonds tie proteins together, with alpha carbons bound to NH. There are 20 amino acids in 4 groups: nonpolar, polar/uncharged, negatively charged/acidic, positively charged/basic. Two structural motifs of proteins are alpha helixes, which look like a ribbon or coil, and beta sheets, in which two or more parallel strands are joined by noncovalent hydrogen bonds. The second half of the lecture was spent showing how to use a protein rendering program.
Video 6, More About Receptors as Proteins, considers more aspects of receptors, taking nicotinic acetylcholine receptor as an example. NAR is made of 5 similar subunits and has a cytosolic region and membrane region composed mainly of alpha helixes and an extracellular binding region made of beta sheets. A ligand binds at an aromatic box, which is composed in NAR of 5 aromatic amino acid residues (tyrosine and tryptophan for NAR). The aromatic box is an interface between two of the five subunits and (1) allows nicotine bonding and (2) is formed by amino acid residues of more than one subunit. Each of the 5 NAR subunits has four transmembrane portions and an extramembrane binding domain. Next, the question of how drug binding produces changes to activate receptor proteins is answered. Drug binding produces conformational changes such as swivels that are allosteric. Most receptors are allosteric proteins (Greek: "other body"), which bind a protein at one site but change function and conformation at another site. Sometimes, all subunits of a ligand-gated channel undergo concerted transitions between 2 or more states, such as open, closed, or desensitized. At other times, sequential transitions occur, meaning that the ligand induces a fit of the protein. Allosteric proteins are also known as shape shifting proteins. We are still unable to accurately predict the structure of proteins, especially membrane proteins, based on their sequence alone,but increases in computational power and experience are moving us in that direction.
Video 7, Introduction to Mammalian Brains: Neuronal circuits, neurons, and synapses, begins with the anatomical terms rostral (front), dorsal (top), caudal (back), and ventral (bottom). Function is often localized in different regions of the brain, with motor and sensory cortex near top, vision in the back, coordination in the basal ganglion and cerebellum, memory in the hippocampus, and the reward pathway, which is important for many brain-acting drugs (e.g., nicotine, dopamine). The spinal cord contains ascending and descending neurons. Spinal reflexes do not involve the brain and sometimes need only two neurons, a sensory neuron wrapped around special muscle fibers to serve as a strain gauge and a motor nerve to cause muscle movement (as well as a motor nerve going to the opposite muscle to inhibit contraction). A neuron contains many parts, which include presynaptic terminal and postsynaptic dendrite, dendrites (Greek: tree), axon (Greek: axis, the central part), myelin sheath wrapping the axon, and synapse (Greek: connection, junction). Synapses were discovered with electron micrography in the 1940s. The human brain contains 10^11 neurons, each with 10^3 synapses. Synapses are formal connections between two neurons. Information flow is one way and occurs when signalling molecules (neurotransmitters) are released from the presynaptic cell, cross the 500-Angstrom synaptic cleft, and are received by receptors in the postsynaptic dendrite.
Video 8, Sample Recordings and Techniques for Studying the Brain presents several videos showing action potentials of nerves stimulated with electricity and drugs. The key point is "The major language of the nervous system is the frequency of a neuron's action potentials." In the first experimental setup, in which a microelectrode is inserted into a nerve, the application of current increases firing frequency. In a similar setup, the application of acetylcholine increases firing frequency, meaning that drugs can affect firing frequency. Many techniques, such as MEG+ERP, PET, and fMRI, are noninvasive ways to image neuronal activity. The resolution, time frame, and invasiveness of the techniques vary.
Video 9, Botulinum Toxin, gives an overview of botulinum toxin, which Clostridium botulinum synthesizes as a single chain (1296 AA) that is cleaved by a protease between 448 and 449, but a disulfide bond at cysteine residues holds the two fragments together. They enter a neuron, which has a reducing environment that results in disulfide bond cleavage, allowing the light chain to act as an enzyme that prevents synaptic transmission. BT is used therapeutically for gastroparesis (delayed gastric emytyping), hemiplegia, blepharospasm, glabellar lines, etc. BT cannot cross the blood brain barrier because it is a large protein.
Video 10, Origin of the Resting Potential, uses several mathematical formulas to quantify current/ion flows in nerves. Nature stores energy in concentration gradients without osmotic stress by having low internal Na+ and high internal K+, but sums are the same, so the osmotic pressure is equilibrated. Cells have K+ channels permeable to potassium, which diffuses down the K+ gradient (outward) with lost positive charge, leading to net negative interior potential.
Video 11, Electrical Aspects of Ion Channels, illustrates ion channels as conductors through a membrane. As there are many per cell, they conduct in parallel when open. Microscopic conductance is the conductance through one, macroscopic conductance is the sum of all microscopic conductance. Kirchhoff's current law (conservation of charge) requires K+ outflow for every Na+ inflow, so Na/K flow in loop, the charge movement across the membrane is conserved, and K+ outflow is balanced by Na+ inflow. Ion channels have two major 'roles' in the context of brain-acting drugs: Open in response to drugs at synapses (neurotransmitters/agonists) and open in response to drugs at axons/cell bodies. Neurons rapidly depolarize membrane potential by opening sodium channels in the membrane.
Week 3 Video I-1: Principles and Receptors
First of 4 mini-lectures on the G protein pathway.
Overview: Receptor on cell membrane > G protein activation > effector channel/enzyme > intracellular messenger Ca2+ or cAMP > Kinase > Phosphorylated protein > Enters nucleus to effect transcription
Otto Loewe in 1921 proved chemical synaptic transmission. Connected two frog hearts to Ringer's solution. When first heart stimulated, stopped beating, little while longer second heart stopped beating SO concluded there is a diffusible substance moving from first to second heart. Later found to be to be acetylcholine, acting on muscarinic ACh receptor.
Many postsynaptic membranes have G protein-coupled receptors: G protein-coupled (1) serotonin, (2) muscarinic/ACh, and (3) dopamine receptors.
Several small molecule xmitters are agonists for both ligang-gated and GPCRs:
Xmitter > Ligand-gated channel / GPCR
ACh > nicotinic Ach R / muscarinic ACh R
GABA > GABAA / GABAB
glutamate
serotonin
histamine
dopamine
In-video quiz: Signal transduction in synaptic GPCRs (all that apply) A. Is initiated by the binding of neuroxmitters such as ACh and glutamate D. Can activate intracellular kinases
Plasma membrane components of the G protein pathway: Receptor (with 7 transmembrane domains) is connected to G protein inside membrane. On neuroxmitter or hormone binding, G protein is activated with GTP binding, changing shape. The alpha subunit of the G protein dissociates from the seven-helix receptor and moves to and activates an effector enzyme or channel. This stops when the alpha subunit of GTP is hydrolyzed into GDP and phosphate.
In-video quiz: events occurring during G-protein coupled receptor signal transduction: (1) GTP is hydrolyzed into GDP and phosphate, (2) G-proteins bind to and activate the effector proteins, (3) G-proteins dissociate from the 7-helix receptor, and (4) agonist binds to the 7-helix receptor. Which of the following numeric sequences correctly describes the order of these events? 4321
Generalizations about GPCRs (1) All have 7 helixes, (2) there are about 1000 GPCRs in genome (most are still orphans in that ligands are unknown), (3) Individual GPCRs respond to (a) low-molecular weight neuroxmitter, (b) a short protein such as endorphin, (c) a relatively insoluble lipid such as anndamide (endocannabinoid), (d) an olfactory stimulus, and (e) light in the eye.
What is selective advantage of this complex pathway? Response not directly influenced by neuroxmitter or hormone in terms of chemistry, speed, or localization, all of which are decoupled. But this amplification and indirect coupling requires energy and limits speed and cooperativity.
Week 3 Video I-2: Inhibitory G Proteins and Their Effectors
All G proteins have alpha, beta, and gamma subunits. They are heterotrimeric. Alpha subunit has GDP bound to put it in inactive state. Beta subunit looks like propeller, prevents alpha and beta from interacting with effector.
In-video quiz: Which GP subunit binds GDP? The alpha subunit
In Gi (inhibitory G) protein effector. Patch clamp method using pipette takes a bit of membrane, with compartmentalization (area inside pipette does not interact with that outside). When Gi is placed into patch clamp and purified Gβγ subnit, potassium channels open and close SO G potassum channels are an effector.
Adding K+ channels keep the membrane potential from reaching the threshold, decreasing the firing rate.
In-video quiz: Inhibitory GPs such as Gi inhibit neuronal firing by binding to and: b. Opening K+ channels.
Gi inhibits neurons (slow neuronal firing/decrease synaptic transmitter release) by (1) directly activating some K+ channels, (2) directly inhibiting some voltage-gated Ca2+ channels, and (3) directly inhibiting adenylyl cyclase.
Week 3 Video I-3: The Stimulatory G Protein (Gs) Pathway
Gs effector is enzyme called cyclase, which coverts ATP to cyclic AMP. Caffeine prolongs intracellular messenger cAMP, phosphodiesterase breaks diester bond of cAMP, producing AMP, but caffeine inhibits this. Generalize by saying phosphodiesterase inhibitors prolong the life of intracellular messengers eg cAMP.
In-video quiz: The enzyme phosphodiesterase: b. Can be inhibited by caffeine
Further downstream, cAMP binds to kinase (protein kinase A), which consists of a regulatory subunit and inactive catalytic subunits. The activated catalytic subunits are released and phosphorylate serine residues in proteins, changing their function. Phosphatases reverse this chemical reaction.
In-video quiz: What type of enzyme can add a phosphate group to the serine residues in a protein? Kinase
Example of effects of cAMP pathway on β-adrenergic receptors (which regulate accommodation in hippocampal neurons). Normally, glutamate pulse in hippocampal neuron depolarizes cell, causing spikes. Ca2+ inflow causes repolarization, stopping firing. But norepinephrine-induced phosphorylation inhibits small-conductance Ca2+ activated potassium channel (SK). The neuron can continue firing. But washing away norepinephrine returned the cell to a normal firing pattern with accommodation. Similar firing when 8-bromo cAMP (which is not hydrolyzed by phosphodiesterase) and forskolin (which activates cyclase) in presence of tetrodotoxin.
Week 3 Video I-4: The Ca-mobilizing G Protein (Gq) pathway: Modifying G protein pathways
The Gq pathway leads to enzyme effector that produces intracellular messengers including Ca2+. Lots of steps: GTP causes separation of phospholipase C-β to separate from G protein α subunit. Causes separation of phosphatidyl inositol 4,5 bisphosphate embedded in membrane and inositol 1,4,5-tirphosphate (IP3), which opens IP3-gated Ca2+ release channel in endoplasmic reticulum, which releases Ca2+ into the cytosol from the ER lumen. The results are (1) Ca2+ binds to and activated protein kinase C and Ca2+ becomes intracellular messenger, activating other enzymes.
In-video quiz: The G-protein Gq increases intracellular calcium because it: (c) Triggers calcium release from the endoplasmic reticulum into the cytosol.
Discussion of all the genes and enzymes associated with G proteins.
In-video quiz: G-protein coupled receptors: (b) Can modulate the function of enzymes and ion channels and (c) can affect intracellular calcium and cAMP levels
Discussion of regulators of G protein signaling (RGS), which tune the kinetics of effector activation/deactivation
In-video quiz: RGS proteins alter the time course of the cardiac muscarinic response because the:
Week 3 Video I-5: Outside-in Mechanisms for Long-term Actions on G Protein Pathways
Focus on nucleus. Electricity is short term (seconds/minutes) language of nervous system. Seymour Benzer (Caltech nobel laurate) uses apparatus with odorant and electric shocks to demonstrate learning in Drosophila. A dunce mutant was isolated that couldn’t learn. These mutants had phosphodiesterase mutations that rendered phosphodiesterase non-functional (i.e., cannot convert cAMP to AMP). Another mutant, rutabaga, lied around like vegetables. This mutant had cyclase mutation (i.e., cannot convert ATP to cAMP).
Transcription factors in nucleus are phosphorylated (e.g., Ca2+ responsive element binder (CREB)). pCREB binds to CRE to prompt transcription of activated target gene.
In-video quiz: cAMP promotes gene transcription by binding to: Protein kinase A
This is outside-in control. Receptor > G protein Gi/Gq/Gs/Gt > Effector (channel or enzyme) > Intracellular messenger (Ca2+/cAMP) > Kinase > Phosphorylated protein > Transcription (takes 10 s to days, but messages can travel up to 1 m to activate genes).
In-video quiz: The outside-in pathway: (A) Leads to alterations in gene expression, (B) Is initiated by agonist binding to G-protein coupled receptors, and (C) Involves the movement of cytoplasmic proteins through nuclear pores.
Week 3 Video II-1: Neurotransmitter Transporters 1
Focus on Raphe nuclei (rostral and caudal system).
There are 3 classes of proteins that transport ions across membranes (1) Ion channels and (2) Ion-coupled transporters (both passive) and (3) Ion pumps, which are active. Ion-coupled transporters transport small molecules across membrane using gradient established by ion pumps.
Neurotransmitter entry into presynaptic terminals: Na+-coupled cell membrane neurotransmitter transporters use Na+ gradient (larger concn outside) to transport small organic molecules (eg, serotonin, dopamine) into cell.
In-video quiz: Serotonin and dopamine are transported across the plasma membrane by: Carrier-mediated transport.
After entry, serotonin and dopamine are brought by H+-coupled vesicular neurotransmitter transporters. These vesicles, which have ATP-driven proton pumps and proton-coupled neurotransmitter transporters to take dopamine/serotonin into vesicle, contain 1000 to 10,000 molecules of the neurotransmitter and ATP.
In-video quiz: Neurotransmitters are transported across the synaptic vesicle membrane using the: Proton gradient.
Plasma membrane neurotransmitter transporters are more susceptible to drugs because they are on the outside of the cell. Examples of neurotransmitter transporter genes are SERT (serotonin), DAT (dopamine), and NET (norephinephrine/noradrenaline. Plasma membrane neurotransmitter transporters have two main jobs (1) terminate neuroxmitter action and (2) replenish neuroxmitter in presynaptic terminal for loading into synaptic vesicles,
MDMA, or ecstasy, works by entering neuroxmitter vesicles and taking protons out, thereby shutting down proton gradient. The vesicle empties of neuroxmitter.
In-video quiz: Amphetamines such as MDMA: (A) Deplete neuroxmitters from synaptic vesicles, (B) Deplete proton gradient across the vesicular membrane, and (D) Cause slow neuroxmitter release into the synaptic cleft.
Na+ coupled cell membrane serotonin transporters are major target for antidepressants (SSRIs) and drugs of abuse (MDMA), Na+ coupled cell membrane dopamine transporters are target for ADHD, amphetamines, cocaine
Week 3 Video II-2: Neurotransmitter transporters as molecules
Alternating access model for ion-coupled transporters: Transmitter transporter can face outside or inside compartment. Transporters wait for molecule from outside to bind, undergoes conformational change, and releases molecule to inside compartment, then flips back. Can do this via symport (transported molecule with co-transported ion) or antiport molecule in, ion out). Also uniport allows molecules in with no ion flow.
In-video quiz: Alternating access model of neuroxmitter transport states: (C) Access to the extracellular and intracellular compartments alternates.
Ion-coupled transporters are like ion channels with gates at both ends. Gate rules: Open when molecules are bound, then other gate opens. Sometimes both gates mistakenly open, turning transporter into ion channel. SSRIs can get transporter stuck in a certain part of the cycle.
In-video quiz: SSRIs: (B) Block serotonin re-uptake and (C) Bind to and stabilize an intermediate state of the serotonin transporter
GAT1 (GABA) transporter inhibited by anticonvulsant tiagabine. This prolongs GABA lifetime, thereby decreasing excess neuronal firing. GABA transporters normall clear all 10,000 transmitter molecules from single synaptic vesicle in few ms.
GFP linked to GABA transporter to determine GAT1 density in different parts of mice brain. Presynaptic fibers of Purkinje cells stains heavily. Density is 1000 per square micron.
Week 3 Video III-1: Recreational Drugs, Overview
Rec drugs typically begin working in a few minutes and work generally for several hours. Related to synapses, channel blockers, G protein pathways, etc.
Examples of rec drugs (which are not necessarily addictive, abused, or illegal) are morphine, tetrahydrocannabinol, ethanol, caffeine, S-ketamine, LSD, nicotine, cocaine, and amphetamine. These have about 500 MW and generally have nitrogen atom(s). Penetrate rapidly into brain.
In-video quiz: Which of the following recreational drugs are amines: (b) cocaine and (c) LSD (ethanol and tetrahydrocannabinol are not).
Most rec drugs come from plants (e.g., cocaine from leaves of coca plant, morphine/heroin from poppies, amphetamine is synthetic but based on plant compounds, LSD is synthetic but based on ergot (wheat fungus), ketamine is synthetic not based on any natural drugs).
Acid-base chemistry of chemistry. Cocaine base (freebase) is uncharged, treatment with HCl gives cocaine HCl, a readily soluble salt. Cocaine enters lungs/nose/stomach as neutral molecule that can permeate > blood > CSF. It is in equilibrium between neutral and protonated cocaine in blood and CSF.
In-video quiz: Mixing cocaine with calcium hydroxide enhances the effect of the drug because: (c) Deprotonates the amine group of cocaine and (d) increases the rate of drug permeation through lipid membranes
Routes into body: Eat/drink, inhale, smoke/vape, inject. Most do not need to be eaten.
Week 3 Video III-2: Targets
Mostly receptors, transporters, and channels.
(1) Neuroxmitter transporters targeted by amphetamine and cocaine
(2) Ligand-activated channels targeted by ketamine and nicotine
(3) Enzymes targeted by caffeine (intracellular target)
(4) GPCRs targeted by LSD, morphine/heroin, and tetrahydrocannabinol
Caffeine is inhibitor of cAMP. Morphone/heroin is agonist of endorphins, ketamine is antagonist
In-video quiz: The endogenous ligand for morphine receptors is: (D) Endorphin.
Morphine/heroin targets GPCR (Gi), μ-opioid receptor; THC targets GPCR (Gi); nicotine targets agonist-activated channel, cocaine targets plasma membrane neuroxmitter xporter; amphetamine targets vesicular & plasma membrane neuroxmitter xporter; ethanol may target K channel; LSD targets CPCR (Gq); caffeine acts on enzyme (cAMP phosphodiesterase)
In-video quiz: Which of the following rec drugs bind to GPCRs? (A) Morphine
How do we know targets? Use knockout mice. Hypothesize response requires a target molecule and knock out gene of molecule, replace with GFP. Then measure drug response in knockout vs. wild-type mice. Knockout mice cannot always be made (sometimes loss of genes results in death at birth).
In-video quiz: Which of the following behaviors would dopamine transporter knockout mice display? (A) Hyperactivity and (C) Diminished cocaine response
Most rec drugs (ex alcohol) at at ≤ 10-5 M.
Week 3 Video III-3: System-level Effects
Look beyond effects at neurons. First look at dopaminergic neurons (centered in nucleus accumbens and ventral tegmental area) Noradrenergic neurons less common, mostly in locus ceruleus (= blue area). Noradrenalin regulates flight or flight response.
In-video quiz: Dopaminergic neurons involved in the reward pathway are located in the: (D) Ventral tegmental area and perhaps substansia nigra.
Serotenergic neurons concentrated in raphe nuclei in brainstem.
System level actions (dopamine pleasure system, noradrenaline readiness system, also perception-association system and decreased neuronal activity). See slide.
fMRI measurements on hallucinogenic 5-HT2A agonist in human brain: psilocybin from mushrooms. Neuronal activity decreased in anterior and posterior cingulate and thalamus.
Rec drugs can have varying overall effects: Inhibitory, excitatory, or dissociation
In-video quiz: Drugs such as LSD and psilocybin induce hallucinations by binding to: (A) Serotonin receptors
Video 4, Drug Entry into the Nervous System, discusses routes into the body, such as lungs (smoking), chewing, patches, injection, suppositories, and application in creams. Drugs such as alkaloids enter the body and diffuse through membranes in neutral form (RNH2) but act on receptor in charged form (RNH3+). Ammonium hydroxide is added to some cigarettes to keep nicotine neutral so more permeates to the brain. Parkinson's disease is treated with levodopa, a prodrug (drug that is converted into another drug in the body) that is a zwitterion (contains both positive and negative groups) that is converted by carboxylases into dopamine in the brain. Dopamine cannot cross the blood brain barrier (BBB) but once in the brain is taken up by dopaminergic neurons for use. The BBB consists of capillaries with tight junctions found only in the brain (holes are present in other parts of the body) that zip endothelial cells together so that only certain nonpolar and hydrophobic molecules can pass, via the endothelial cell membranes.
Video 5, Introduction to Drug Receptors...A very basic lecture: A receptor is a molecule on the cell surface or interior with affinity for a drug or ligand (Greek: to tie). Most receptors are proteins. Amide or peptide bonds tie proteins together, with alpha carbons bound to NH. There are 20 amino acids in 4 groups: nonpolar, polar/uncharged, negatively charged/acidic, positively charged/basic. Two structural motifs of proteins are alpha helixes, which look like a ribbon or coil, and beta sheets, in which two or more parallel strands are joined by noncovalent hydrogen bonds. The second half of the lecture was spent showing how to use a protein rendering program.
Video 6, More About Receptors as Proteins, considers more aspects of receptors, taking nicotinic acetylcholine receptor as an example. NAR is made of 5 similar subunits and has a cytosolic region and membrane region composed mainly of alpha helixes and an extracellular binding region made of beta sheets. A ligand binds at an aromatic box, which is composed in NAR of 5 aromatic amino acid residues (tyrosine and tryptophan for NAR). The aromatic box is an interface between two of the five subunits and (1) allows nicotine bonding and (2) is formed by amino acid residues of more than one subunit. Each of the 5 NAR subunits has four transmembrane portions and an extramembrane binding domain. Next, the question of how drug binding produces changes to activate receptor proteins is answered. Drug binding produces conformational changes such as swivels that are allosteric. Most receptors are allosteric proteins (Greek: "other body"), which bind a protein at one site but change function and conformation at another site. Sometimes, all subunits of a ligand-gated channel undergo concerted transitions between 2 or more states, such as open, closed, or desensitized. At other times, sequential transitions occur, meaning that the ligand induces a fit of the protein. Allosteric proteins are also known as shape shifting proteins. We are still unable to accurately predict the structure of proteins, especially membrane proteins, based on their sequence alone,but increases in computational power and experience are moving us in that direction.
Video 7, Introduction to Mammalian Brains: Neuronal circuits, neurons, and synapses, begins with the anatomical terms rostral (front), dorsal (top), caudal (back), and ventral (bottom). Function is often localized in different regions of the brain, with motor and sensory cortex near top, vision in the back, coordination in the basal ganglion and cerebellum, memory in the hippocampus, and the reward pathway, which is important for many brain-acting drugs (e.g., nicotine, dopamine). The spinal cord contains ascending and descending neurons. Spinal reflexes do not involve the brain and sometimes need only two neurons, a sensory neuron wrapped around special muscle fibers to serve as a strain gauge and a motor nerve to cause muscle movement (as well as a motor nerve going to the opposite muscle to inhibit contraction). A neuron contains many parts, which include presynaptic terminal and postsynaptic dendrite, dendrites (Greek: tree), axon (Greek: axis, the central part), myelin sheath wrapping the axon, and synapse (Greek: connection, junction). Synapses were discovered with electron micrography in the 1940s. The human brain contains 10^11 neurons, each with 10^3 synapses. Synapses are formal connections between two neurons. Information flow is one way and occurs when signalling molecules (neurotransmitters) are released from the presynaptic cell, cross the 500-Angstrom synaptic cleft, and are received by receptors in the postsynaptic dendrite.
Video 8, Sample Recordings and Techniques for Studying the Brain presents several videos showing action potentials of nerves stimulated with electricity and drugs. The key point is "The major language of the nervous system is the frequency of a neuron's action potentials." In the first experimental setup, in which a microelectrode is inserted into a nerve, the application of current increases firing frequency. In a similar setup, the application of acetylcholine increases firing frequency, meaning that drugs can affect firing frequency. Many techniques, such as MEG+ERP, PET, and fMRI, are noninvasive ways to image neuronal activity. The resolution, time frame, and invasiveness of the techniques vary.
Video 9, Botulinum Toxin, gives an overview of botulinum toxin, which Clostridium botulinum synthesizes as a single chain (1296 AA) that is cleaved by a protease between 448 and 449, but a disulfide bond at cysteine residues holds the two fragments together. They enter a neuron, which has a reducing environment that results in disulfide bond cleavage, allowing the light chain to act as an enzyme that prevents synaptic transmission. BT is used therapeutically for gastroparesis (delayed gastric emytyping), hemiplegia, blepharospasm, glabellar lines, etc. BT cannot cross the blood brain barrier because it is a large protein.
Video 10, Origin of the Resting Potential, uses several mathematical formulas to quantify current/ion flows in nerves. Nature stores energy in concentration gradients without osmotic stress by having low internal Na+ and high internal K+, but sums are the same, so the osmotic pressure is equilibrated. Cells have K+ channels permeable to potassium, which diffuses down the K+ gradient (outward) with lost positive charge, leading to net negative interior potential.
Video 11, Electrical Aspects of Ion Channels, illustrates ion channels as conductors through a membrane. As there are many per cell, they conduct in parallel when open. Microscopic conductance is the conductance through one, macroscopic conductance is the sum of all microscopic conductance. Kirchhoff's current law (conservation of charge) requires K+ outflow for every Na+ inflow, so Na/K flow in loop, the charge movement across the membrane is conserved, and K+ outflow is balanced by Na+ inflow. Ion channels have two major 'roles' in the context of brain-acting drugs: Open in response to drugs at synapses (neurotransmitters/agonists) and open in response to drugs at axons/cell bodies. Neurons rapidly depolarize membrane potential by opening sodium channels in the membrane.
Week 3 Video I-1: Principles and Receptors
First of 4 mini-lectures on the G protein pathway.
Overview: Receptor on cell membrane > G protein activation > effector channel/enzyme > intracellular messenger Ca2+ or cAMP > Kinase > Phosphorylated protein > Enters nucleus to effect transcription
Otto Loewe in 1921 proved chemical synaptic transmission. Connected two frog hearts to Ringer's solution. When first heart stimulated, stopped beating, little while longer second heart stopped beating SO concluded there is a diffusible substance moving from first to second heart. Later found to be to be acetylcholine, acting on muscarinic ACh receptor.
Many postsynaptic membranes have G protein-coupled receptors: G protein-coupled (1) serotonin, (2) muscarinic/ACh, and (3) dopamine receptors.
Several small molecule xmitters are agonists for both ligang-gated and GPCRs:
Xmitter > Ligand-gated channel / GPCR
ACh > nicotinic Ach R / muscarinic ACh R
GABA > GABAA / GABAB
glutamate
serotonin
histamine
dopamine
In-video quiz: Signal transduction in synaptic GPCRs (all that apply) A. Is initiated by the binding of neuroxmitters such as ACh and glutamate D. Can activate intracellular kinases
Plasma membrane components of the G protein pathway: Receptor (with 7 transmembrane domains) is connected to G protein inside membrane. On neuroxmitter or hormone binding, G protein is activated with GTP binding, changing shape. The alpha subunit of the G protein dissociates from the seven-helix receptor and moves to and activates an effector enzyme or channel. This stops when the alpha subunit of GTP is hydrolyzed into GDP and phosphate.
In-video quiz: events occurring during G-protein coupled receptor signal transduction: (1) GTP is hydrolyzed into GDP and phosphate, (2) G-proteins bind to and activate the effector proteins, (3) G-proteins dissociate from the 7-helix receptor, and (4) agonist binds to the 7-helix receptor. Which of the following numeric sequences correctly describes the order of these events? 4321
Generalizations about GPCRs (1) All have 7 helixes, (2) there are about 1000 GPCRs in genome (most are still orphans in that ligands are unknown), (3) Individual GPCRs respond to (a) low-molecular weight neuroxmitter, (b) a short protein such as endorphin, (c) a relatively insoluble lipid such as anndamide (endocannabinoid), (d) an olfactory stimulus, and (e) light in the eye.
What is selective advantage of this complex pathway? Response not directly influenced by neuroxmitter or hormone in terms of chemistry, speed, or localization, all of which are decoupled. But this amplification and indirect coupling requires energy and limits speed and cooperativity.
Week 3 Video I-2: Inhibitory G Proteins and Their Effectors
All G proteins have alpha, beta, and gamma subunits. They are heterotrimeric. Alpha subunit has GDP bound to put it in inactive state. Beta subunit looks like propeller, prevents alpha and beta from interacting with effector.
In-video quiz: Which GP subunit binds GDP? The alpha subunit
In Gi (inhibitory G) protein effector. Patch clamp method using pipette takes a bit of membrane, with compartmentalization (area inside pipette does not interact with that outside). When Gi is placed into patch clamp and purified Gβγ subnit, potassium channels open and close SO G potassum channels are an effector.
Adding K+ channels keep the membrane potential from reaching the threshold, decreasing the firing rate.
In-video quiz: Inhibitory GPs such as Gi inhibit neuronal firing by binding to and: b. Opening K+ channels.
Gi inhibits neurons (slow neuronal firing/decrease synaptic transmitter release) by (1) directly activating some K+ channels, (2) directly inhibiting some voltage-gated Ca2+ channels, and (3) directly inhibiting adenylyl cyclase.
Week 3 Video I-3: The Stimulatory G Protein (Gs) Pathway
Gs effector is enzyme called cyclase, which coverts ATP to cyclic AMP. Caffeine prolongs intracellular messenger cAMP, phosphodiesterase breaks diester bond of cAMP, producing AMP, but caffeine inhibits this. Generalize by saying phosphodiesterase inhibitors prolong the life of intracellular messengers eg cAMP.
In-video quiz: The enzyme phosphodiesterase: b. Can be inhibited by caffeine
Further downstream, cAMP binds to kinase (protein kinase A), which consists of a regulatory subunit and inactive catalytic subunits. The activated catalytic subunits are released and phosphorylate serine residues in proteins, changing their function. Phosphatases reverse this chemical reaction.
In-video quiz: What type of enzyme can add a phosphate group to the serine residues in a protein? Kinase
Example of effects of cAMP pathway on β-adrenergic receptors (which regulate accommodation in hippocampal neurons). Normally, glutamate pulse in hippocampal neuron depolarizes cell, causing spikes. Ca2+ inflow causes repolarization, stopping firing. But norepinephrine-induced phosphorylation inhibits small-conductance Ca2+ activated potassium channel (SK). The neuron can continue firing. But washing away norepinephrine returned the cell to a normal firing pattern with accommodation. Similar firing when 8-bromo cAMP (which is not hydrolyzed by phosphodiesterase) and forskolin (which activates cyclase) in presence of tetrodotoxin.
Week 3 Video I-4: The Ca-mobilizing G Protein (Gq) pathway: Modifying G protein pathways
The Gq pathway leads to enzyme effector that produces intracellular messengers including Ca2+. Lots of steps: GTP causes separation of phospholipase C-β to separate from G protein α subunit. Causes separation of phosphatidyl inositol 4,5 bisphosphate embedded in membrane and inositol 1,4,5-tirphosphate (IP3), which opens IP3-gated Ca2+ release channel in endoplasmic reticulum, which releases Ca2+ into the cytosol from the ER lumen. The results are (1) Ca2+ binds to and activated protein kinase C and Ca2+ becomes intracellular messenger, activating other enzymes.
In-video quiz: The G-protein Gq increases intracellular calcium because it: (c) Triggers calcium release from the endoplasmic reticulum into the cytosol.
Discussion of all the genes and enzymes associated with G proteins.
In-video quiz: G-protein coupled receptors: (b) Can modulate the function of enzymes and ion channels and (c) can affect intracellular calcium and cAMP levels
Discussion of regulators of G protein signaling (RGS), which tune the kinetics of effector activation/deactivation
In-video quiz: RGS proteins alter the time course of the cardiac muscarinic response because the:
Week 3 Video I-5: Outside-in Mechanisms for Long-term Actions on G Protein Pathways
Focus on nucleus. Electricity is short term (seconds/minutes) language of nervous system. Seymour Benzer (Caltech nobel laurate) uses apparatus with odorant and electric shocks to demonstrate learning in Drosophila. A dunce mutant was isolated that couldn’t learn. These mutants had phosphodiesterase mutations that rendered phosphodiesterase non-functional (i.e., cannot convert cAMP to AMP). Another mutant, rutabaga, lied around like vegetables. This mutant had cyclase mutation (i.e., cannot convert ATP to cAMP).
Transcription factors in nucleus are phosphorylated (e.g., Ca2+ responsive element binder (CREB)). pCREB binds to CRE to prompt transcription of activated target gene.
In-video quiz: cAMP promotes gene transcription by binding to: Protein kinase A
This is outside-in control. Receptor > G protein Gi/Gq/Gs/Gt > Effector (channel or enzyme) > Intracellular messenger (Ca2+/cAMP) > Kinase > Phosphorylated protein > Transcription (takes 10 s to days, but messages can travel up to 1 m to activate genes).
In-video quiz: The outside-in pathway: (A) Leads to alterations in gene expression, (B) Is initiated by agonist binding to G-protein coupled receptors, and (C) Involves the movement of cytoplasmic proteins through nuclear pores.
Week 3 Video II-1: Neurotransmitter Transporters 1
Focus on Raphe nuclei (rostral and caudal system).
There are 3 classes of proteins that transport ions across membranes (1) Ion channels and (2) Ion-coupled transporters (both passive) and (3) Ion pumps, which are active. Ion-coupled transporters transport small molecules across membrane using gradient established by ion pumps.
Neurotransmitter entry into presynaptic terminals: Na+-coupled cell membrane neurotransmitter transporters use Na+ gradient (larger concn outside) to transport small organic molecules (eg, serotonin, dopamine) into cell.
In-video quiz: Serotonin and dopamine are transported across the plasma membrane by: Carrier-mediated transport.
After entry, serotonin and dopamine are brought by H+-coupled vesicular neurotransmitter transporters. These vesicles, which have ATP-driven proton pumps and proton-coupled neurotransmitter transporters to take dopamine/serotonin into vesicle, contain 1000 to 10,000 molecules of the neurotransmitter and ATP.
In-video quiz: Neurotransmitters are transported across the synaptic vesicle membrane using the: Proton gradient.
Plasma membrane neurotransmitter transporters are more susceptible to drugs because they are on the outside of the cell. Examples of neurotransmitter transporter genes are SERT (serotonin), DAT (dopamine), and NET (norephinephrine/noradrenaline. Plasma membrane neurotransmitter transporters have two main jobs (1) terminate neuroxmitter action and (2) replenish neuroxmitter in presynaptic terminal for loading into synaptic vesicles,
MDMA, or ecstasy, works by entering neuroxmitter vesicles and taking protons out, thereby shutting down proton gradient. The vesicle empties of neuroxmitter.
In-video quiz: Amphetamines such as MDMA: (A) Deplete neuroxmitters from synaptic vesicles, (B) Deplete proton gradient across the vesicular membrane, and (D) Cause slow neuroxmitter release into the synaptic cleft.
Na+ coupled cell membrane serotonin transporters are major target for antidepressants (SSRIs) and drugs of abuse (MDMA), Na+ coupled cell membrane dopamine transporters are target for ADHD, amphetamines, cocaine
Week 3 Video II-2: Neurotransmitter transporters as molecules
Alternating access model for ion-coupled transporters: Transmitter transporter can face outside or inside compartment. Transporters wait for molecule from outside to bind, undergoes conformational change, and releases molecule to inside compartment, then flips back. Can do this via symport (transported molecule with co-transported ion) or antiport molecule in, ion out). Also uniport allows molecules in with no ion flow.
In-video quiz: Alternating access model of neuroxmitter transport states: (C) Access to the extracellular and intracellular compartments alternates.
Ion-coupled transporters are like ion channels with gates at both ends. Gate rules: Open when molecules are bound, then other gate opens. Sometimes both gates mistakenly open, turning transporter into ion channel. SSRIs can get transporter stuck in a certain part of the cycle.
In-video quiz: SSRIs: (B) Block serotonin re-uptake and (C) Bind to and stabilize an intermediate state of the serotonin transporter
GAT1 (GABA) transporter inhibited by anticonvulsant tiagabine. This prolongs GABA lifetime, thereby decreasing excess neuronal firing. GABA transporters normall clear all 10,000 transmitter molecules from single synaptic vesicle in few ms.
GFP linked to GABA transporter to determine GAT1 density in different parts of mice brain. Presynaptic fibers of Purkinje cells stains heavily. Density is 1000 per square micron.
Week 3 Video III-1: Recreational Drugs, Overview
Rec drugs typically begin working in a few minutes and work generally for several hours. Related to synapses, channel blockers, G protein pathways, etc.
Examples of rec drugs (which are not necessarily addictive, abused, or illegal) are morphine, tetrahydrocannabinol, ethanol, caffeine, S-ketamine, LSD, nicotine, cocaine, and amphetamine. These have about 500 MW and generally have nitrogen atom(s). Penetrate rapidly into brain.
In-video quiz: Which of the following recreational drugs are amines: (b) cocaine and (c) LSD (ethanol and tetrahydrocannabinol are not).
Most rec drugs come from plants (e.g., cocaine from leaves of coca plant, morphine/heroin from poppies, amphetamine is synthetic but based on plant compounds, LSD is synthetic but based on ergot (wheat fungus), ketamine is synthetic not based on any natural drugs).
Acid-base chemistry of chemistry. Cocaine base (freebase) is uncharged, treatment with HCl gives cocaine HCl, a readily soluble salt. Cocaine enters lungs/nose/stomach as neutral molecule that can permeate > blood > CSF. It is in equilibrium between neutral and protonated cocaine in blood and CSF.
In-video quiz: Mixing cocaine with calcium hydroxide enhances the effect of the drug because: (c) Deprotonates the amine group of cocaine and (d) increases the rate of drug permeation through lipid membranes
Routes into body: Eat/drink, inhale, smoke/vape, inject. Most do not need to be eaten.
Week 3 Video III-2: Targets
Mostly receptors, transporters, and channels.
(1) Neuroxmitter transporters targeted by amphetamine and cocaine
(2) Ligand-activated channels targeted by ketamine and nicotine
(3) Enzymes targeted by caffeine (intracellular target)
(4) GPCRs targeted by LSD, morphine/heroin, and tetrahydrocannabinol
Caffeine is inhibitor of cAMP. Morphone/heroin is agonist of endorphins, ketamine is antagonist
In-video quiz: The endogenous ligand for morphine receptors is: (D) Endorphin.
Morphine/heroin targets GPCR (Gi), μ-opioid receptor; THC targets GPCR (Gi); nicotine targets agonist-activated channel, cocaine targets plasma membrane neuroxmitter xporter; amphetamine targets vesicular & plasma membrane neuroxmitter xporter; ethanol may target K channel; LSD targets CPCR (Gq); caffeine acts on enzyme (cAMP phosphodiesterase)
In-video quiz: Which of the following rec drugs bind to GPCRs? (A) Morphine
How do we know targets? Use knockout mice. Hypothesize response requires a target molecule and knock out gene of molecule, replace with GFP. Then measure drug response in knockout vs. wild-type mice. Knockout mice cannot always be made (sometimes loss of genes results in death at birth).
In-video quiz: Which of the following behaviors would dopamine transporter knockout mice display? (A) Hyperactivity and (C) Diminished cocaine response
Most rec drugs (ex alcohol) at at ≤ 10-5 M.
Week 3 Video III-3: System-level Effects
Look beyond effects at neurons. First look at dopaminergic neurons (centered in nucleus accumbens and ventral tegmental area) Noradrenergic neurons less common, mostly in locus ceruleus (= blue area). Noradrenalin regulates flight or flight response.
In-video quiz: Dopaminergic neurons involved in the reward pathway are located in the: (D) Ventral tegmental area and perhaps substansia nigra.
Serotenergic neurons concentrated in raphe nuclei in brainstem.
System level actions (dopamine pleasure system, noradrenaline readiness system, also perception-association system and decreased neuronal activity). See slide.
fMRI measurements on hallucinogenic 5-HT2A agonist in human brain: psilocybin from mushrooms. Neuronal activity decreased in anterior and posterior cingulate and thalamus.
Rec drugs can have varying overall effects: Inhibitory, excitatory, or dissociation
In-video quiz: Drugs such as LSD and psilocybin induce hallucinations by binding to: (A) Serotonin receptors
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