Chapter 1-8 Neurotransmitter Signaling: Key Terms

Context and setup

• Speaker recap: Jeremy discussing glutamate and broader neurotransmitter systems; personal note about hibernation research and inviting feedback at Neurocritical Care Society meeting in Montreal (clinical-scientist audience; few PhDs).
• Administrative updates: sheet updated to include an extra grad student; Perusall platform used for automatic grading of comments; students can see their grade as they work; balance between class discussion and online scoring.
• Goals of today: review the paper on fatty acid receptors (FFARs), link to prior lipid metabolism discussions, and revisit GPCR concepts, including Arrestin, biased agonism, and receptor signaling nuances.
• Emphasis: a mix of conceptual understanding, practical interpretation of curves, and clinical/pharmacological implications.

Neurotransmitter systems overview (non-glutamatergic examples mentioned)

• ATP as a neurotransmitter
  • ATP is stored in vesicles and released as a transmitter; excitatory role.
  • ATP is also a precursor to adenosine; adenosine acts as a neuromodulator.
• Adenosine and its receptors
  • Four adenosine receptor subtypes highlighted: A1, A2A, A2B, A3.
  • Receptors show differential potency and efficacy for the same downstream readout (e.g., cyclic AMP production).
• Endocannabinoid system
  • Synthesized on demand, not stored in vesicles; released from postsynaptic terminal; neuromodulatory role with retrograde signaling characteristics.
• Gaseous neurotransmitters
  • Nitric oxide (NO) and carbon monoxide (CO) discussed as neuromodulators; not stored in vesicles; diffuse quickly.

Fatty acid receptors paper: main ideas and context

• Relevance to lipids and metabolism
  • Free fatty acids act on G protein-coupled receptors (GPCRs); insulin signaling and inflammation contexts discussed (e.g., omega-3s from fish oil).
• Terminology review from the paper
  • Reiteration of GPCR basics: transmembrane seven-helix structure; some prefer calling them GPCRs rather than “transmembrane seven receptors.”
  • Metabolic context and receptor pharmacology tied to lipid signaling.
• Conceptual takeaway from curves in the paper
  • Dose-response curves shown beyond what class discussed previously; different curves illustrate how endogenous agonists interact with a full agonist to shape responses.

Dose-response curves: walkthrough and interpretation

• Baseline concept
  • Measure response (y-axis) to drug concentration (x-axis, log scale).
  • A full agonist can drive a system to maximal response; endogenous agonist can further modulate response depending on receptor occupancy.
• Key curve concepts
  • Full agonist + endogenous agonist co-incubation shifts the curve visually; darker color indicates higher full-agonist concentration.
  • Partial agonist: starting with a partial response; additional endogenous agonist can increase the response but may appear as antagonism relative to the partial agonist alone.
  • Competitive (surmountable) antagonist: presence of antagonist shifts the curve to the right (requires more agonist to achieve same effect).
  • Positive allosteric modulators (PAMs): leftward shift of the dose-response curve; enhance endogenous agonist effect at the same orthosteric site.
  • Agonist plus PAM that also acts as partial agonist at high concentrations: complex modulation that can resemble the full agonist effect at high allosteric engagement.
  • Superagonist: elicits greater-than-typical maximal response under certain conditions; may produce a rightward shift in the presence of endogenous agonist due to competition dynamics.
• Antagonists and their effects
  • Classic antagonist: zero efficacy; blocks response without producing a direct effect.
  • Noncompetitive antagonist: reduces maximal response, not easily overcome by increasing orthosteric agonist.
  • Inverse agonist: reduces baseline constitutive activity below basal, illustrating constitutive receptor activity in the absence of ligand.
• Practical takeaway
  • The curve illustrates what competition, efficacy, and allosteric modulation do to receptor signaling and observed responses.
  • The same receptor can display different functional outcomes depending on downstream coupling and cellular context (signal transduction pathways).

Receptor theory concepts: affinity, occupancy, efficacy, and their relationships

• Definitions and distinctions
  • Affinity: strength of binding between receptor and ligand; quantified by KD (dissociation constant).
  • Occupancy: fraction of receptors bound by ligand; depends on ligand concentration and KD.
  • Efficacy: ability of ligand-receptor occupancy to produce a cellular response; not solely determined by binding.
  • EC50: concentration of ligand that produces 50% of the maximal effect; not necessarily equal to KD due to downstream signaling and receptor coupling nuances.
• Key relationships
  • A ligand can have high affinity (low KD) but low efficacy (partial agonist); conversely, high efficacy does not guarantee high affinity.
  • biologically, occupancy does not always translate linearly to response because of signal amplification, receptor reserve, and pathway bias.
• Quantitative concepts to remember
  • Receptor occupancy for a ligand with concentration [L] and KD: ext{Occupancy} = rac{[L]}{[L] + K_D}
  • Simple sigmoidal dose-response form (Emax model): E = E{ ext{max}} rac{[A]}{[A] + EC{50}}
  • For receptor occupancy vs. efficacy, a common model introduces an efficacy parameter: E = E{ ext{max}} rac{ au [A]}{ au [A] + KA} where au represents efficacy and K_A is affinity; higher au yields greater efficacy at a given occupancy.
• Practical implication
  • KD and EC50 diverge when receptor reserve or biased signaling is present; a high-affinity ligand may exhibit less apparent efficacy if downstream signaling is limited or decoupled.

KD vs Ki and selectivity across receptors

• Distinct constants
  • KD: affinity of a ligand for a receptor (binding affinity).
  • Ki: inhibition constant for a competitor in competitive binding assays; reflects how effectively an antagonist blocks binding.
• Selectivity and measurement
  • Tables (Table 1 and Table 2 in the paper) show how drugs bind to various 5-HT receptor subtypes and serotonin transporters; measured in nanomolar ranges (Ki or KD values).
  • A rule of thumb discussed: aim for roughly two orders of magnitude difference in affinity to claim selectivity, especially when curves are plotted on a log scale. This helps ensure the shifts in dose-response are meaningful and separable.
• Practical note
  • In physiology, closely related receptor subtypes may have very similar affinities; functional selectivity (bias) and receptor context often drive observed effects more than raw affinity alone.

Autoreceptors and somatodendritic autoreceptors (with 5-HT1A example)

• What autoreceptors do
  • Autoreceptors provide negative feedback to the neuron that releases the neurotransmitter; they regulate firing rate and transmitter release.
• Somatodendritic autoreceptors
  • Located on the soma and dendrites; for serotonin, the 5-HT1A somatodendritic autoreceptor reduces neuronal firing when activated by serotonin or a 5-HT1A agonist.
  • Example: buspirone is a 5-HT1A agonist used as an anxiolytic and to suppress shivering via dorsal raphe modulation.
• Postsynaptic autoreceptors
  • There can be autoreceptors at presynaptic terminals as well that modulate transmitter release.
• Specific case: 5-HT1A autoreceptors
  • Activation slows firing of serotonergic neurons in the dorsal raphe; this reduces overall serotonin release.
• Conceptual takeaway
  • Presynaptic autoreceptors (somatodendritic or terminal) provide feedback control of neurotransmitter output; their activation can paradoxically reduce network activity even though they are receptors for the same transmitter.
  • Buspirone as an example: 5-HT1A agonist that dampens dorsal raphe firing and thereby reduces serotonin release, with clinical anxiolytic effects.

Biased signaling and arrestin pathways in GPCR pharmacology

• What is biased signaling?
  • Some ligands preferentially activate one signaling pathway over another through the same receptor (e.g., G protein vs. beta-arrestin pathways).
  • Two illustrative receptor contexts: unbiased agonist (similar effects on G protein and arrestin) vs biased agonist (selective activation of one pathway).
• Arrestin’s dual role
  • G protein signaling: classic rapid signal transduction via G proteins (Gs, Gi/o, Gq, etc.).
  • Arrestin recruitment: upon receptor phosphorylation, beta-arrestin can bind, initiating arrestin-mediated signaling and receptor internalization via clathrin-coated pits.
• Consequences of arrestin signaling
  • Internalization and receptor desensitization/tolerance: prolonged or repeated agonist exposure leads to receptor endocytosis and reduced surface receptor availability.
  • Arrestin-related signaling pathways influence cell survival, growth, and other kinase cascades beyond G protein signaling.
• Implications for drug development
  • Biased agonism can be exploited to achieve therapeutic effects with fewer side effects by avoiding detrimental G protein pathways and leveraging arrestin-mediated outcomes (or vice versa).
• Internalization and tolerance example
  • Receptors are pulled into the cell (endocytosis) via clathrin-coated pits; this results in rapid tolerance to certain GPCR agonists when administered repeatedly.
• Summary from the talk
  • Arrestin is not only about receptor internalization; it also mediates distinct intracellular signaling that can have diverse biological consequences.

Allosteric modulation and biased allosteric modulation (BAMS)

• Allosteric modulators (AMs)
  • Bind at sites distinct from the orthosteric (genuine binding) pocket.
  • Positive allosteric modulators (PAMs): enhance the effect of the endogenous ligand; can shift the dose-response curve left and increase maximal response in the presence of the endogenous ligand.
  • Negative allosteric modulators (NAMs): diminish the effect of the endogenous ligand; can shift curves right or reduce maximal response when efficacy is present.
• Biased allosteric modulators (BAMS)
  • Allosteric modulators that preferentially bias signaling toward G protein or arrestin pathways, thereby shaping downstream responses in a pathway-specific way.
• Practical takeaway from the slides
  • Allosteric sites provide a mechanism to finely tune receptor signaling without directly competing with the endogenous ligand at the orthosteric site.
  • The concept of BAMS is part of a broader strategy to achieve pathway-selective pharmacology with potentially improved safety profiles.

Biochemical signaling and receptor-state concepts: two-state and occupancy–efficacy views

• Two-state model and occupancy–efficacy coupling
  • Receptors exist in active and inactive conformations; ligand binding shifts the equilibrium toward one state, altering downstream signaling.
  • The functional response (E) depends on both receptor occupancy and the efficacy of the bound ligand in a given signaling context.
• Constitutive activity and inverse agonism
  • Some receptors exhibit baseline activity even without ligand (constitutive activity).
  • Inverse agonists can reduce this baseline activity below basal levels; neutral antagonists block signaling without changing basal activity.
• Receptor reserve (spare receptors)
  • Maximum response can be achieved without occupying all receptors; a small fraction of receptor occupancy can achieve full efficacy due to signal amplification.
  • Implication: EC50 can be much lower than KD; a ligand may produce near-maximal effect with submaximal receptor occupancy if a receptor reserve exists.
• Relevance to therapeutic pharmacology
  • Receptor reserve and signaling bias help explain why drugs with similar affinities can have very different clinical effects.

Receptor subtype specifics and signaling outcomes (examples mentioned in the talk)

• Adenosine receptors (A1, A2A, A2B, A3)
  • Different receptor subtypes show varying potency and efficacy for cyclic AMP production and other readouts.
  • A1 and A2A have distinct roles in neuromodulation and are differentially activated by endogenous adenosine.
• Dopamine receptors
  • D1 and D5 (D1-like): generally couple to Gs/olf (activate adenylyl cyclase).
  • D2, D3, D4 (D2-like): couple to Gi/o (inhibit adenylyl cyclase).
  • Functional outcomes are tissue-specific due to receptor coupling and downstream signaling context.
• 5-HT (serotonin) receptors and autoreceptors
  • 5-HT1A as an autoreceptor on somatodendritic sites reduces firing when activated; 5-HT1A agonists include buspirone (anxiolytic).
  • Somatodendritic autoreceptors are a key example of how receptor location affects function (firing rate vs. postsynaptic responses).
• Summary implication
  • Receptor subtypes can have very different signaling fingerprints; exact outcomes depend on receptor location, coupling, and downstream signaling networks.

Methods to study receptor signaling and neurotransmission (techniques discussed)

• GRAB receptors (biased allosteric sensors)
  • Genetically encoded fluorescent sensors that report endogenous ligand binding via conformational changes.
  • Techniques include FRET-based designs and circularly permuted GFP variants that fluoresce upon receptor activation.
  • Applicability: broad, enables real-time monitoring of receptor activation by endogenous ligands.
• Receptor internalization and arrestin pathways (conceptual links to GRAB sensors)
  • Arrestin recruitment is linked to receptor internalization and to arrestin-mediated signaling pathways beyond G protein signaling.
• Measuring neurotransmitters in vivo: microdialysis and electrochemical methods
  • Microdialysis: semipermeable membrane catheter in brain tissue; sampled extracellular fluid diffuses across membrane; can quantify neurotransmitters via LC-MS; good for concentration changes but limited temporal resolution (milliseconds not achieved).
  • Electrochemical amperometry/fast-scan cyclic voltammetry (FSCV): detect oxidation currents from electroactive neurotransmitters (e.g., dopamine, serotonin) at microelectrodes; highly time-resolved but less straightforward for absolute quantification; good for relative changes.
  • The combination of microdialysis with LC-MS and electrochemical methods advanced the ability to quantify neurotransmitter dynamics with varying temporal and spectral resolutions.
• Practical considerations
  • GRAB sensors and biased signaling concepts provide a modern toolkit to dissect receptor-specific signaling in real time.
  • The choice of method depends on whether absolute concentrations, temporal resolution, or pathway-specific signaling is of interest.

Practical/philosophical implications and real-world relevance

• Drug development and therapeutic targeting
  • Understanding bias (G protein vs arrestin) and allosteric modulation enables design of drugs with targeted signaling outcomes and fewer side effects.
  • Autoreceptor biology informs strategies to modulate transmitter release and neuronal firing in disorders such as anxiety, depression, and movement disorders.
• Research context and cross-disciplinary relevance
  • Interplay between lipid signaling (FFARs), neurotransmitter dynamics, and receptor pharmacology has implications for neurology, psychiatry, and pharmacology.
  • Tools like GRAB sensors, BAMS, and advanced measurement techniques open new avenues to understand in vivo signaling in health and disease.

Quick takeaways and quiz-style prompts from the talk

• Define somatodendritic autoreceptor and name the typical autoreceptor subtype for 5-HT in this context.
  • Answer guidance: somatodendritic autoreceptor; 5-HT1A autoreceptor.
• Explain how receptor affinity (KD) and functional affinity (EC50) can diverge in systems with receptor reserve or bias.
• Describe how a competitive antagonist shifts dose-response curves and how Schild analysis can be used to quantify affinity.
• Distinguish PAMs from NAMs and give an example of how each would alter a dose-response curve in the presence of endogenous ligand.
• Describe the concept of biased agonism and give an example of how arrestin-biased signaling differs from G-protein signaling in terms of downstream outcomes.
• Explain how receptor internalization (via arrestin and clathrin-pit endocytosis) contributes to pharmacological tolerance.
• Briefly outline microdialysis and FSCV as complementary methods for measuring neurotransmitter dynamics in vivo, including a key limitation of each.
• What is a GRAB receptor, and why is it useful for studying endogenous ligand dynamics?

Notes on structure and formatting

• All equations and quantitative relations are presented in LaTeX, enclosed in double dollar signs, e.g. E = E{ ext{max}} rac{[A]}{[A] + EC{50}}.
• The notes are organized as top-level headings with detailed bullet-point content underneath, mirroring a comprehensive study guide that could replace the original source.

Closing reminders from the talk

• There will be a scheduled Brain Energy Atlas session on the 29th; potential workshop on MATLAB integration with data analysis, dengan a focus on practical data handling.
• The discussion included reflections on using LLMs for coding assistance and the evolving role of computational tools in neuroscience research.
• Attendees were encouraged to verify AI-generated code and to approach tools like LLMs as assistants rather than substitutes for rigorous scientific reasoning.