Chapter 3 Notes: Signaling Across Synapses

Chapter 3 Notes: Signaling across Synapses

3.1 Arrival of the action potential at the presynaptic terminal triggers neurotransmitter release

All trillions of synapses in our brains work essentially the same way as the neuromuscular junction.
Neurotransmitters: molecules released by presynaptic neurons that diffuse across the synaptic cleft and act on postsynaptic targets.
Vertebrate NMJ (neuromuscular junction) as a model synapse: motor neuron terminals release acetylcholine (ACh).
NMJ identified ACh in the 1930s; postsynaptic muscle fiber is giant, enabling intracellular recording of synaptic transmission (end-plate potential, EPP).
Historical context: Otto Loewi and Henry Dale were awarded the Nobel Prize for discovering chemical transmission. Loewi's famous 'Vagusstoff' experiment demonstrated that stimulating the vagus nerve of one frog heart released a chemical (later identified as ACh) that could slow the heart rate of a second, isolated frog heart when its perfusing fluid was transferred.
Motor axon terminals form hundreds of sites that release ACh onto the muscle fiber, making NMJ a robust converter of action potentials to muscle contraction.
Each muscle fiber is innervated by only one motor neuron, unlike brain neurons which can receive thousands of inputs (e.g., $10,000$ to $100,000$ synapses). Motor axon terminals at the NMJ release $\sim7000$ ACh molecules per vesicle, contributing to a high safety factor and reliable muscle contraction upon nerve firing. In vertebrates, NMJ inputs are exclusively excitatory, contrasting with some invertebrates where both excitatory and inhibitory axons can innervate the same muscle.
Experimental setups often used conditions that prevent muscle contraction to avoid movement artifacts.

Classic experiments establish: action potentials in motor axon trigger ACh release, which binds muscle ACh receptors and depolarizes muscle, producing EPPs.
Early experiments showed that ACh application via micropipette (iontophoresis) could evoke EPPs, and blocking axonal action potentials with tetrodotoxin (TTX) prevented nerve-stimulated EPPs but not ACh-evoked EPPs, supporting the idea that APs trigger transmitter release rather than directly depolarizing the postsynaptic cell.
Fusion of synaptic vesicles with presynaptic membrane releases discrete packets of ACh; this led to the quantal view of neurotransmission.

3.2 Neurotransmitters are released in discrete packets (quanta)

Bernard Katz and colleagues observed miniature end-plate potentials (mEPPs) in muscle fibers in the absence of nerve stimulation. This quantal release was established before electron microscopes could visually confirm synaptic vesicles.
mEPPs have a unitary size, suggesting a basic unit of transmission. The 'quantum amplitude' (Q) refers to the postsynaptic response elicited by a single vesicle's worth of neurotransmitter.
Evoked EPPs under normal conditions appear as the sum of many mEPPs (unit quanta).

Quantal hypothesis: neurotransmitters are released in discrete packets (quanta) of relatively uniform size. The overall amplitude of the evoked postsynaptic response at a synapse can be described as the product of three factors: the number of release sites (N), the probability of release at each site (P), and the quantum amplitude (Q).
Ca2+ dependence and quantal content: reducing extracellular Ca2+ lowers release probability; evoked EPPs under low Ca2+ are often equal in amplitude to mEPPs, sometimes $2-3\times$ unit size when multiple quanta release.
Statistical testing of quantal release:
- If k quanta are released per stimulus, the frequency follows a Poisson distribution with mean m (m = mean number of quanta per stimulus):
  $f(k; \text{Poisson}, m) = \frac{m^k e^{-m}}{k!}$
- Relationship to experiments: the frequency of synaptic failures (k = 0) and occurrences of $1\times$ , $2\times$ , $3\times$ mEPPs matched Poisson predictions when m was determined from the ratio of mean EPP amplitude to mean mEPP amplitude.

Poisson assumptions and NMJ context: large n (hundreds of release sites) and small p (probability per quantum) satisfy Poisson criteria; CNS synapses may not always meet Poisson assumptions due to smaller n or nonuniform p.
Binomial distribution note (Box 3-1): if there are n quanta with release probability p, the distribution is f(k;n,p) = \frac{n!}{k!(n-k)!} p^k (1-p)^{n-k}$
- When n is large and p is small, the binomial distribution can be approximated by the Poisson distribution with parameter
 \lambda = np $</li><li>$ \lambda $can be estimated experimentally as the mean number of quanta released per stimulus, equal to the ratio of the mean EPP amplitude to the mean mEPP amplitude:$ \lambda \text{ (or } m) = \frac{\text{mean EPP amplitude}}{\text{mean mEPP amplitude}} $</li></ul></li></ul><ul><li>Key assumptions for Poisson estimation: small p, large n, independent release of quanta, uniform p across quanta, and relatively uniform vesicle content. CNS synapses may violate these assumptions; some CNS synapses with single active zones may be better described by a binomial model with n between$ 1 $and$ 10 $. Experimentally, determining both N (number of release sites) and P (probability of release) for the binomial distribution is challenging due to difficulties in precisely identifying the number of release sites and measuring non-uniform release probabilities.</li></ul>3.3 Neurotransmitters are released when synaptic vesicles fuse with the presynaptic plasma membrane<ul><li>Electron microscopy (EM) revealed$ \sim40 $nm diameter synaptic vesicles in presynaptic terminals, often docked near the presynaptic membrane at active zones. Flash-freezing techniques captured vesicles in the act of exocytosis.</li><li>The uniform vesicle size aligns with the concept of uniform quantal size (e.g., frog NMJ$ \sim7000 $ACh molecules per vesicle).</li><li>Vesicle fusion with the plasma membrane releases neurotransmitter into the cleft, producing a miniature postsynaptic depolarization (mEPP) or larger responses when multiple vesicles fuse.</li><li>NMJ has high quantal content (hundreds of vesicles released per action potential) vs. many CNS synapses with much lower quantal content.</li><li>Across chemical synapses, basic structural elements are conserved: docked vesicles at active zones; postsynaptic densities opposite active zones; vesicles and active zone composition conserved across species. EM observations pre- and post-stimulation support vesicle fusion as the mechanism for transmitter release. Electron microscopy typically depicts vesicles as empty membrane bags; however, electron tomography reveals synaptic vesicles as crowded protein machines with proteins drawn to scale, suggesting high protein density often obscured by fixation conditions or electron scattering properties in conventional EM.</li></ul>3.4 Neurotransmitter release is controlled by Ca2+ entry into the presynaptic terminal<ul><li>Ca2+ influx is required for action-potential-triggered transmitter release; low extracellular Ca2+ reduces release probability.</li><li>Key experimental evidence from the squid giant synapse shows: depolarization opens presynaptic voltage-gated Ca2+ channels, Ca2+ enters, and postsynaptic currents follow. A tail current after depolarization indicates Ca2+ entry and subsequent transmitter release. The tail current experiment specifically highlights that some of the latency in transmitter release is due to the inherent time required for voltage-gated Ca2+ channels to open.</li><li>Ca2+ entry timing is tightly coupled to release: brief Ca2+ influx during depolarization triggers vesicle fusion near active zones. The release process itself shows a highly nonlinear, cooperative dependence on Ca2+ concentration, well-modeled by 5 Ca2+ binding sites (akin to oxygen binding to hemoglobin) on the release machinery.</li><li>Two experimental approaches demonstrate Ca2+ dependence:<ul><li>Local Ca2+ uncaging or 'caged Ca2+' experiments show that an instantaneous rise in Ca2+ triggers release even without depolarization.</li><li>Calcium-sensitive dyes reveal localized Ca2+ elevations at active zones during stimulation, consistent with microdomain signaling.</li></ul></li></ul><ul><li>Sequenced events (frog NMJ and squid giant synapse): Action potential$ \to $Presynaptic depolarization$ \to $Opening of voltage-gated Ca2+ channels$ \to $Ca2+ entry$ \to $Fusion of docked vesicles with presynaptic membrane$ \to $Neurotransmitter release.</li><li>Two contributing factors to rapid release: a ready-to-fuse (docked) vesicle pool and a Ca2+-triggered conformational change in fusion machinery that occurs extremely fast, with minimal ATP-dependent catalysis at the final fusion step.</li><li>The Calyx of Held, a large synapse in the auditory system, serves as an experimental model to study rapid synaptic transmission, demonstrating$ \sim1 $ms latency between pre- and postsynaptic action potentials, crucial for quick responses in processes like prey capture or predator evasion. The speed and locality of Ca2+-triggered release are critically governed by diffusion constraints: Ca2+ diffusion is very fast over short distances (e.g.,$ \sim10 $nm in microseconds) but significantly slower over longer distances (e.g., 1 micron takes$ \sim10 $milliseconds). This necessitates that Ca2+ channels are anchored extremely close to vesicle release sites to ensure rapid, localized signaling without significant temporal delays or spatial spillover.</li></ul>3.5 SNARE proteins mediate synaptic vesicle fusion<ul><li>Core SNARE components drive fusion:<ul><li>v-SNARE: synaptobrevin/VAMP (vesicle-associated)</li><li>t-SNARE: syntaxin (plasma membrane)</li><li>t-SNARE: SNAP-25 (plasma membrane, lipid-anchored)</li></ul></li></ul><ul><li>SNAREs assemble into a tight four-helix bundle (two from SNAP-25 and syntaxin, one from synaptobrevin) that forms a zipper-like structure, pulling vesicle and plasma membranes together to drive fusion. The zipper proceeds from the distal ends toward the membrane, releasing energy to fuse bilayers.</li><li>The SNARE complex architecture: a four-helix bundle with synaptobrevin, syntaxin, and SNAP-25 contributing helices; the transmembrane portions are not part of the crystal structure but anchor the complex.</li><li>SM protein: Munc18 (Sec1/Munc18 family) binds SNAREs and is essential for fusion in mammals. The SM protein Munc18 (mammalian uncoordinated-18) and Unc13 (another associated protein) were identified through studies of 'uncoordinated' (unc) gene mutations in C. elegans, initially isolated by Sydney Brenner, highlighting the evolutionary conservation of vesicle trafficking machinery.</li><li>Structural basis of fusion and toxins: many proteases cleave SNAREs, blocking fusion and transmitter release; SNAREs are central to vesicle fusion in many intracellular trafficking steps beyond synaptic release.</li><li>The SNARE mechanism is shared broadly in intracellular fusion events, with functional SNAREs and SM proteins forming the minimal fusion machinery. In 2013, Randy Schekman, James Rothman, and Thomas Südhof were awarded the Nobel Prize for their discoveries of machinery regulating vesicle traffic, including the SNARE components crucial for synaptic release.</li></ul>3.6 Synaptotagmin acts as a Ca2+ sensor to trigger synaptic vesicle fusion<ul><li>Synaptotagmin (Syt), especially Syt-1, on synaptic vesicles contains multiple Ca2+-binding sites and acts as the primary Ca2+ sensor that triggers fusion. Synaptotagmin features two C2 domains (C2A and C2B) that cooperatively bind Ca2+ ions (e.g., 3 and 2 Ca2+ ions respectively), triggering interactions with the SNARE complex to facilitate fusion.</li><li>Genetic evidence: knockout of Syt-1 reduces synaptic transmission. While Syt-1 knockout demonstrates its necessity for transmission, knock-in experiments with Syt-1 mutations that reduce Ca2+ binding affinity (e.g., causing half as much Ca2+ binding) provide stronger evidence that Syt-1 is the direct Ca2+ sensor, as such mutations predictably reduce release.</li><li>Complexin: a regulatory protein that both activates the SNARE complex and blocks it at an intermediate step; proposed model: Ca2+-bound synaptotagmin relieves the complexin block to allow SNARE-mediated fusion.</li><li>Fast CNS synapses show extremely short latency from Ca2+ entry to postsynaptic response (e.g.,$ \sim150\mu\text{s} $to postsynaptic depolarization in fast mammalian CNS synapses), consistent with a primed vesicle pool and Ca2+-triggered fusion.</li><li>In summary: action potential$ \to $presynaptic depolarization$ \to $opening of Ca2+ channels$ \to $Ca2+ entry$ \to $fusion of primed vesicles$ \to $neurotransmitter release.</li><li>Ca2+ microdomains near active zones enable cooperative binding to synaptotagmin and rapid, localized fusion. Paradoxically, synaptotagmin's relatively low affinity and fast off-rate for Ca2+ binding are essential. This ensures that Ca2+ binding is highly local to the active calcium microdomains, allowing rapid termination of release and preventing unwanted spatial or temporal overlap between release sites.</li></ul>3.7 The presynaptic active zone is a highly organized structure<ul><li>The active zone coordinates fast neurotransmitter release by bringing docked vesicles into close proximity with Ca2+ channels.</li><li>Core active zone components:<ul><li>Unc13: promotes assembly of SNAREs and tethers vesicles to the release site.</li><li>RIM and RIM-BP: recruit and organize Ca2+ channels near synaptic vesicles; interact with Rab3 to position vesicles at the active zone.</li><li>Rab3 on vesicles interacts with active-zone proteins; connections to cytoskeleton help vesicle trafficking.</li></ul></li></ul><ul><li>Trans-synaptic adhesion molecules align the presynaptic active zone with postsynaptic densities: neurexin (presynaptic) and neuroligin (postsynaptic) and cadherins connect pre- and postsynaptic membranes. Adhesion molecules like cadherins are ultimately attached to the cytoskeleton of the pre-synaptic and post-synaptic cells.</li><li>The active zone is linked to a broader scaffold that includes other proteins and the actin cytoskeleton; recent super-resolution studies reveal detailed organization around Ca2+ channel clusters and Bruchpilot/ELKS scaffolds in model organisms.</li><li>This architecture ensures that Ca2+ influx efficiently triggers fusion at closely apposed release sites and aligns with postsynaptic receptor-rich zones.</li></ul>3.8 Neurotransmitter clearance from the synaptic cleft<ul><li>Rapid clearance is essential for continued signaling and avoiding spillover.</li><li>Acetylcholine at NMJ is rapidly degraded by acetylcholinesterase in the cleft, ensuring quick termination of ACh signaling.</li><li>Poisons (e.g., nerve gases like sarin, insecticides) block acetylcholinesterase, leading to ACh buildup, hyperactivation of receptors, and subsequent paralysis due to inactivation of postsynaptic Na+ channels. The paralysis occurs due to prolonged depolarization of the postsynaptic membrane by ACh buildup, which inactivates voltage-gated Na+ channels in the muscle fiber, rendering them unable to fire action potentials and inhibiting muscle contraction. Choline is reuptaken into the presynaptic terminal for ACh resynthesis.</li><li>In many CNS systems, neurotransmitters are cleared by reuptake via plasma membrane transporters (symporters using Na+ gradients) back into presynaptic cytosol or glia; vesicular transporters in synaptic vesicles then repackage neurotransmitter using a proton gradient.</li><li>Neurotransmitter uptake into synaptic vesicles is driven by a vesicular ATPase (V-ATPase) that acidifies the vesicle's lumen by pumping protons inward. This creates an electrochemical proton gradient, which is then utilized by specific proton-coupled vesicular neurotransmitter transporters to import neurotransmitter molecules into the vesicle.</li><li>In glutamatergic synapses, glial uptake can also contribute to clearance.</li><li>Reuptake and recycling are important targets for psychiatric drugs. Selective Serotonin Reuptake Inhibitors (SSRIs), common antidepressants, function by blocking serotonin reuptake transporters, increasing serotonin concentration in the synaptic cleft. The full mechanism by which this leads to antidepressant effects is still under investigation, implying complex downstream signaling.</li></ul>3.9 Synaptic vesicle recycling by endocytosis is essential for continual synaptic transmission<ul><li>After exocytosis, vesicle membranes and proteins must be retrieved and regenerated.</li><li>Three main pathways for vesicle membrane retrieval:1) Kiss-and-run: transient fusion with limited exchange of membrane proteins/lipids, vesicle reseals and is recycled. The 'kiss-and-run' mechanism, proposing transient vesicle fusion without full collapse, remains a subject of significant debate among cellular neuroscientists, with some evidence supporting and other evidence questioning its prevalence or existence under normal conditions.2) Clathrin-mediated endocytosis: vesicle membrane fully collapses into the presynaptic membrane and is retrieved via clathrin-coated pits.3) Ultrasonic/ultrafast endocytosis: rapidly forms large endocytic vesicles near the active zone, which then become endosomes and are converted into synaptic vesicles via clathrin-mediated steps. Ultrasonic/ultrafast endocytosis involves the rapid formation of large endocytic vesicles that internalize substantial membrane portions near the active zone. These larger vesicles then mature into endosomes, from which new synaptic vesicles are generated via clathrin-mediated budding.</li><li>Sorting of vesicle components involves NSF disassembling SNARE complexes after fusion; syntaxin and SNAP-25 remain in the plasma membrane, synaptobrevin returns to the vesicle; other vesicle proteins are retrieved via adaptor proteins.</li><li>Dynamin (Shibire in Drosophila) is essential for vesicle scission in endocytosis; temperature-sensitive Shibire mutations block endocytosis, depleting vesicle pools and blocking transmission. When dynamin function is abolished, this process stops, and many vesicles get stuck during retrieval, rapidly depleting vesicle pools, leading to a cessation of release and severe animal dysfunction or death.</li><li>Readily releasable pool and vesicle recycling efficiency are critical for sustained signaling. The readily releasable pool refers to vesicles that are not immediately competent for release but can be quickly primed and docked at the active zone for rapid exocytosis. This pool can be measured by stimulating the synapse until all available vesicles are depleted.</li><li>Vesicle recycling efficiency can be studied experimentally by carefully measuring changes in the capacitance of the presynaptic membrane, which reflects the addition and retrieval of membrane area during exocytosis and endocytosis.</li></ul>3.10 Synapses can be facilitating or depressing<ul><li>Synaptic efficacy is modified by prior activity (short-term plasticity): facilitation or depression during trains of action potentials. The flexibility to adjust synaptic strength is crucial for learning and memory.</li><li>Facilitation: successive action potentials trigger larger postsynaptic responses because Ca2+ accumulates in the presynaptic terminal, increasing release probability. Facilitation occurs when successive action potentials lead to a buildup of residual Ca2+ in the presynaptic terminal, which, when combined with new Ca2+ influx, significantly increases the probability of neurotransmitter release for subsequent stimuli.</li><li>Depression: initial high release probability depletes the readily releasable pool, reducing transmission with ongoing activity; recovery can occur within seconds as vesicles are replenished. Depression results from the depletion of the readily releasable vesicle pool due to high initial release probability, leading to a reduction in subsequent neurotransmitter release until vesicles can be replenished.</li><li>The same synapse can show facilitation or depression depending on intrinsic properties and recent activity. Synapses with high initial release probability tend to show depression (e.g., for detecting transient events), while those with low initial probability show facilitation (e.g., for detecting prolonged input). This varied short-term plasticity allows synapses to differentially encode information based on the temporal patterns of activity.</li></ul>3.11 Nervous systems use many neurotransmitters<ul><li>The NMJ uses acetylcholine (ACh); the CNS uses glutamate for excitation and GABA/glycine for inhibition; monoamines (serotonin, dopamine, norepinephrine, histamine) modulate activity; neuropeptides act as modulators and can be co-released with small-molecule transmitters.</li><li>Glutamate is the major CNS excitatory transmitter; GABA (and glycine in the brainstem/spinal cord) are major inhibitory transmitters.</li><li>Historically, Dale's Law posited that each neuron releases only one type of neurotransmitter at all its synapses; however, this has been disproven. Many neurons exhibit co-transmission, releasing multiple types of neurotransmitters (e.g., glutamate and GABA; dopamine and GABA) either from separate vesicle populations or, less commonly, through co-release of multiple transmitters from the same vesicles (e.g., GABA and glycine, or GABA and dopamine). The diverse types of vesicles can be segregated within the same terminal or in different terminals, and their release can have varying Ca2+ sensitivities, allowing for complex modulation of neuronal activity.</li><li>Neuropeptides are released from large dense-core vesicles and often act over longer timescales; they are transported from soma to terminals and are less readily recycled locally.</li><li>Receptors determine transmitter effects: ions and signaling pathways differ across receptor types.</li><li>Table 3-2 lists widely used transmitters (Acetylcholine, Glutamate, GABA, Glycine, Serotonin, Dopamine, Norepinephrine, Histamine, ATP, Neuropeptides) and their major uses; vertebrates also show neuron- and region-specific transmitter roles.</li></ul>3.12 Acetylcholine opens a nonselective cation channel at the neuromuscular junction<ul><li>An inward current (often drawn downwards on graphs) indicates a net positive charge flowing into the cell, which typically causes depolarization. Exogenous ACh depolarizes muscle membranes most effectively near the NMJ, indicating high ACh receptor density there.</li><li>Voltage-clamp experiments show two-electrode recording: ACh-induced end-plate current (EPC - End-Plate Current, also referred to as EIC) is inward at negative potentials and outward at positive potentials; the I–V curve is nearly linear with a reversal potential near$ 0 $mV. The current produced by neurotransmitter release at the NMJ is called the End-Plate Current (EPC), which generates an End-Plate Potential (EPP).</li><li>In central nervous system neurons, similar concepts apply as excitatory/inhibitory postsynaptic currents (EPSCs/IPSCs) produce excitatory/inhibitory postsynaptic potentials (EPSPs/IPSPs).</li><li>The ACh-activated channels typically remain open for$ 3-4 $milliseconds.</li><li>ACh-activated channels are permeable to Na+ and K+ (and some Ca2+), not anions like Cl−; this yields a net inward current at negative potentials and net outward current at positive potentials.</li><li>The reversal potential being$ \sim0 $mV (not equal to$ E{\text{Na}} $or$ E{\text{K}} $) indicates permeability to multiple cations. The ACh-induced current is a nonselective cation current that depolarizes the muscle, leading to the EPP and muscle contraction when threshold is reached. This channel is permeable to both Na+ and K+, but a greater influx of Na+ occurs compared to K+ efflux. This is because the driving force for Na+ ($ V{\text{m}} - E{\text{Na+}} $) is significantly larger than for K+ ($ V{\text{m}} - E{\text{K+}} $), as the resting membrane potential ($ V{\text{m}} $) is generally far from$ E{\text{Na+}} $but relatively close to$ E_{\text{K+}} $.</li></ul>3.13 Skeletal muscle acetylcholine receptor is a ligand-gated ion channel<ul><li>The skeletal muscle ACh receptor (AChR) is a heteropentamer with$ 5 $subunits: two$ \alpha $, one$ \beta $, one$ \gamma $, one$ \delta $; two ACh binding sites are at the$ \alpha \text{–}\gamma $and$ \alpha \text{–}\delta $interfaces. An agonist is a drug or chemical that activates a receptor, mimicking the effect of the natural ligand. An antagonist is a drug or chemical that binds to and blocks a receptor, thereby inhibiting the action of the natural ligand. Agonists (e.g., nicotine) activate these receptors, while antagonists (e.g., D-tubocurarine/curare) block them, hence they are called nicotinic acetylcholine receptors (nAChR). Antagonists can be competitive (binding to the same site as the natural ligand) or non-competitive (binding to an allosteric site elsewhere on the receptor).</li><li>Co-expression of Torpedo AChR subunits in Xenopus oocytes confers ACh-evoked inward currents, blocked by curare, confirming the receptor as the ion channel. The electric ray, Torpedo, with its modified muscle producing electric shocks, is an incredibly rich source of nicotinic acetylcholine receptors, enabling their biochemical isolation and structural mapping.</li><li>AChR subunit structure: channels have four transmembrane helices per subunit; M2 lines the pore; ACh binding causes rotation of$ \alpha $subunits, conformational change in M2, and opening of the gate.</li><li>The receptor is a prototype ligand-gated ion channel; 3D structure (crystal/electron microscopy) reveals pore-lining M2 helices and transmembrane organization.</li></ul>3.14 Neurotransmitter receptors are either ionotropic or metabotropic<ul><li>Ionotropic receptors are ligand-gated ion channels; they are themselves ion channels that directly open or close upon neurotransmitter binding, mediating rapid ($ \sim $milliseconds) changes in membrane potential. Examples include nicotinic ACh receptors (nAChR), GABAA receptors, glycine receptors, AMPA, NMDA, and P2X receptors. Ionotropic receptors vary in subunit composition; for example, nicotinic ACh, GABAA, and glycine receptors typically have$ 5 $subunits, AMPA and NMDA glutamate receptors have$ 4 $, and some ATP receptors have$ 3 $.</li><li>Metabotropic receptors (G protein-coupled receptors or GPCRs) are not ion channels themselves. Instead, they bind neurotransmitters and initiate intracellular signaling cascades (e.g., via G proteins) that indirectly modulate other ion channels or intracellular processes, leading to slower (tens of milliseconds to seconds) and longer-lasting effects.</li><li>Many transmitters have both ionotropic and metabotropic receptors (e.g., ACh via nAChR and mAChR; glutamate via AMPA/NMDA and mGluRs; GABA via GABAA and GABAB).</li><li>Table 3-3 lists representative ionotropic and metabotropic receptors by transmitter (ACh, Glutamate, GABA, Glycine, Serotonin, Dopamine, Norepinephrine, Histamine, ATP, Neuropeptides), including sensory receptors like rhodopsin in photoreceptors and receptors for olfaction and taste.</li></ul>3.15 AMPA and NMDA glutamate receptors are activated by glutamate under different conditions<ul><li>Ionotropic glutamate receptors mediate fast excitatory transmission; they are cation channels with reversal near$ 0 $mV.</li><li>AMPA receptors: fast Na+/K+ permeable; some subtypes are Ca2+ permeable depending on subunit composition (e.g., GluA2 editing affects Ca2+ permeability).</li><li>NMDA receptors: require binding of glutamate and a co-agonist (glycine) and also require postsynaptic depolarization to relieve Mg2+ block; high Ca2+ permeability; contribute to synaptic plasticity and signaling.</li><li>NMDA receptors act as coincidence detectors: require both presynaptic glutamate release and postsynaptic depolarization to open; crucial for plasticity and activity-dependent wiring.</li><li>AMPA receptors provide initial depolarization to relieve Mg2+ block on nearby NMDARs; both receptors contribute to Ca2+ influx and downstream signaling.</li></ul>3.16 Properties of individual ionotropic glutamate receptors specified by subunit composition<ul><li>All ionotropic glutamate receptors have four subunits with modular domains: extracellular amino-terminus (ATD), ligand-binding domain (LBD), transmembrane domain (M1, M3, M4) and pore-forming M2 loop, and intracellular C-terminus.</li><li>AMPA receptors can form homotetramers or heterotetramers (e.g., GluA1–GluA4). Subunit composition affects Ca2+ permeability; most GluA2-containing receptors are Ca2+-impermeable due to RNA editing; lack of GluA2 or unedited GluA2 yields Ca2+ permeability and inward rectification due to polyamine block.</li><li>NMDA receptors are obligatory heterotetramers composed of two GluN1 (GluN1) and two GluN2 (GluN2A-D) subunits; GluN3 variants can substitute for GluN2 in some neurons. Subunit composition influences conductance, pharmacology, and signaling.</li><li>Different subunits confer distinct properties and developmental regulation; TARPs regulate AMPA receptor trafficking, surface expression, and gating properties.</li><li>Subunit composition and TARPs together shape receptor function and plasticity. A recent study showed that making calcium-permeable AMPA receptors in interneurons calcium-impermeable affected their selectivity to visual stimuli, demonstrating a role in perception.</li></ul>3.17 The postsynaptic density is organized by scaffold proteins<ul><li>The postsynaptic density (PSD) at glutamatergic synapses includes receptors (AMPA/NMDA), trans-synaptic adhesion molecules (neurexin/ neuroligin, cadherin), signaling enzymes (CaMKII), and a network of scaffold proteins (e.g., PSD-95).</li><li>PSD-95 binds NMDA receptor subunits (GluN2), AMPA receptor auxiliary subunits (TARPs), neuroligin, and CaMKII, and also links to other receptors and cytoskeleton.</li><li>PSD scaffolds stabilize receptor localization, regulate trafficking, and coordinate signaling with Ca2+ entry; they can cluster receptors near the active zone to optimize transmission.</li><li>PSD organization may involve liquid–liquid phase separation, aiding self-organization of PSD proteins into functional complexes.</li></ul>3.18 Ionotropic GABA and glycine receptors are Cl− channels that mediate inhibition<ul><li>Inhibitory synapses use Cl− channels (GABA_A and glycine receptors) to hyperpolarize or shunt the postsynaptic membrane.</li><li>In spinal cord and brainstem, IPSPs are mediated largely by GABA*A and glycine receptors; reversal potentials around the Cl− equilibrium potential ($ E_{\text{Cl}} $) yield hyperpolarizing or shunting inhibition depending on the context.</li><li>Inhibition can also manifest as shunting inhibition, where a conductance opens, keeping the membrane potential near resting potential and preventing further depolarization, even if it doesn't cause hyperpolarization directly. Shunting inhibition occurs when activating a Cl− conductance (e.g., via GABA_A or glycine receptors) keeps the membrane potential near or below the resting potential, effectively "shunting" or reducing the impact of excitatory currents and preventing depolarization, even if it doesn't cause active hyperpolarization.</li><li>Classic experiment shows IPSP reversal near$ E{\text{Cl}} $; increasing intracellular Cl− shifts$ E{\text{Cl}} $and alters IPSP direction, confirming Cl− conductance as the primary mechanism.</li><li>Inhibition helps suppress excitatory inputs and prevent firing; GABA_A receptor opening adds conductance that can dampen excitatory EPSPs via shunting inhibition.</li><li>GABA_A receptors are pentamers with multiple subunit compositions; glycine receptors resemble nAChR-like pentamers and also form Cl− channels.</li><li>In developing neurons, intracellular Cl− can be high, making GABA excitatory; maturation lowers intracellular Cl− so GABA becomes inhibitory. This developmental shift is due to changes in chloride transporters, which reduce intracellular Cl- and shift$ E_{\text{Cl}} $from depolarizing (e.g.,$ -30 $mV) to hyperpolarizing (e.g.,$ -70 $mV).</li><li>GABA_B receptors (metabotropic) modulate inhibition via G protein signaling to open K+ channels in some cells.</li></ul>3.19 Metabotropic neurotransmitter receptors trigger G protein cascades<ul><li>Metabotropic receptors belong to the GPCR superfamily; they trigger intracellular signaling via heterotrimeric G proteins (G$ \alpha $, G$ \beta $, G$ \gamma $).</li><li>ACh, glutamate, GABA, dopamine, norepinephrine, serotonin, ATP, neuropeptides, and various sensory receptors are GPCRs.</li><li>GPCRs span the membrane seven times; ligand binding promotes conformational change (R$ \to R$^*$) that binds G\alpha\beta\gamma $; GDP on G$ \alpha $is exchanged for GTP, causing G$ \alpha $-GTP and G$ \beta\gamma $dissociation to signal downstream effectors.</li><li>G$ \alpha $has intrinsic GTPase activity, inactivating itself; G$ \beta\gamma $can regulate effectors directly; termination involves GTP hydrolysis and reassociation of G$ \alpha $and G$ \beta\gamma $.</li></ul>3.20 A GPCR signaling paradigm:$ \beta $-adrenergic receptors activate cAMP as a second messenger<ul><li>$ \beta $-adrenergic receptors activated by epinephrine/norepinephrine couple to Gs; Gs-GTP activates adenylyl cyclase (AC) to produce cAMP from ATP.</li><li>cAMP activates protein kinase A (PKA): regulatory subunits bind cAMP, releasing catalytic subunits that phosphorylate substrates.</li><li>PKA signaling affects diverse targets, including voltage-gated Ca2+ channels and HCN channels, affecting excitability and heart rate (illustrative pathway): norepinephrine$ \to\beta $-adrenergic receptor$ \to $Gs$ \to $AC$ \to $cAMP$ \to $PKA$ \to $targets (e.g., Ca2+ channels, HCN channels).</li><li>This pathway illustrates a general mechanism by which a transmitter can modulate neuronal and cardiac function through second messengers.</li></ul>3.21$ \alpha $and$ \beta\gamma $G protein subunits trigger diverse signaling pathways that alter membrane conductance<ul><li>The human genome encodes multiple G$ \alpha $($ 16 $), G$ \beta $($ 5 $), and G$ \gamma $($ 13 $) subunits; combinations yield diverse GPCR signaling.</li><li>Besides G$ \alpha $s pathways (e.g., AC/cAMP/PKA), other variants (e.g., Gi) inhibit AC, reducing cAMP levels and downstream signaling.</li><li>In postsynaptic compartments, the ultimate effectors are often ion channels (e.g., K+ and Ca2+ channels) that alter membrane potential and excitability.</li><li>Example: norepinephrine activation of$ \beta $-adrenergic receptors increases Ca2+ entry via Ca2+ channels (via cAMP/PKA) and can also activate HCN channels to depolarize pacemaker cells.</li></ul>3.22 Metabotropic receptors can act on the presynaptic terminal to modulate neurotransmitter release<ul><li>Metabotropic receptors can modulate presynaptic release, either autocrine (presynaptic neuron modulates its own release) or heteroreceptor signaling (presynaptic terminal affected by other neurons).</li><li>Example: sympathetic neurons releasing norepinephrine have presynaptic$ \alpha $-adrenergic receptors that inhibit voltage-gated Ca2+ channels via G$ \beta\gamma $, reducing Ca2+ entry and transmitter release (presynaptic inhibition/depression).</li><li>Presynaptic facilitation/inhibition: depends on receptor type, G protein coupling, and downstream signaling; can enhance or suppress transmitter release.</li><li>Presynaptic modulation extends to other transmitter systems and is a key mechanism for short-term plasticity.</li></ul>3.23 GPCR signaling features multiple mechanisms of signal amplification and termination<ul><li>Amplification: a single receptor can activate multiple G proteins, each activating many downstream effectors; for example, cAMP production by AC after Gs activation can lead to multiple PKA activations and broad phosphorylation.</li><li>Termination mechanisms: ligand dissociation, intrinsic GTPase activity of G$ \alpha $, reassociation of G$ \beta\gamma $with G$ \alpha $-GDP, AC deactivation, cAMP breakdown by phosphodiesterases, PKA subunit reassociation, and phosphatases reversing phosphorylation.</li><li>Arrestin-mediated desensitization: GPCR kinases phosphorylate activated receptors, enabling arrestin binding; arrestin blocks G protein coupling and can promote receptor endocytosis, potentially initiating alternate signaling routes.</li></ul>3.24 Postsynaptic signaling to the nucleus; immediate early genes (IEGs) and transcription factors<ul><li>Postsynaptic activity can induce long-term changes by triggering gene expression changes in the nucleus (hours to days).</li><li>Immediate early genes (IEGs) such as Fos, Egr1 are rapidly induced by neuronal activity and often encode transcription factors that regulate downstream gene expression.</li><li>A well-studied example: nicotine/cAMP signaling can drive Fos transcription; CREB (cAMP response element-binding protein) phosphorylation activates transcription of Fos and other genes.</li><li>Calcium signaling from NMDA receptors, voltage-gated Ca2+ channels, IP3 receptors, or Ryanodine receptors can activate Ca2+/CaM-dependent kinases and MAP kinase cascades, leading to CREB phosphorylation and transcriptional regulation.</li><li>BDGF and Arc: growth factors and activity-regulated genes regulate synaptic development and plasticity.</li><li>Epigenetic modifications (DNA methylation, histone modification) and mRNA methylation can also modulate activity-dependent gene expression; mutations in signaling components can contribute to brain disorders.</li></ul>3.25 Dendrites are sophisticated integrative devices<ul><li>Excitatory inputs: integration occurs as EPSPs travel to the axon initial segment where action potentials are initiated; dendritic trees contain many excitatory inputs that create complex integration patterns.</li><li>Passive cable properties (time constant$ \tau $, length constant$ \lambda $) influence signal attenuation with distance from soma.</li><li>Model neurons show that distal inputs produce smaller, slower, broader somatic EPSPs due to attenuation; proximal inputs produce larger, faster EPSPs.</li><li>Neurons receive thousands of excitatory inputs; one EPSP is usually insufficient to reach spike threshold; temporal and spatial summation of multiple EPSPs across dendritic compartments is required.</li><li>Dendritic voltage-gated Na+ and Ca2+ channels can amplify EPSPs, potentially creating dendritic spikes that propagate and interact with somatic spikes.</li><li>Back-propagating action potentials (bAPs) from the soma can invade dendrites and interact with EPSPs, possibly enhancing coincidence detection and plasticity (e.g., enabling dendritic spikes when an AP occurs near synaptic input).</li><li>The integration of inputs is compartmentalized by dendritic morphology; different compartments may show distinct excitability and plasticity properties.</li></ul>3.26 Synapses are strategically placed at specific locations in postsynaptic neurons<ul><li>Excitatory synapses are mainly on dendritic spines distributed across the dendritic tree; each spine typically receives input from a single excitatory terminal and acts as a semi-isolated functional unit. The neck of a dendritic spine acts as an electrical and biochemical compartment, providing resistance and limiting molecular diffusion.</li><li>Inhibitory synapses target dendritic spines, shafts, the cell body, and the axon initial segment; this allows targeted modulation of excitatory integration and spike initiation. Unlike excitatory synapses, inhibitory synapses primarily form directly onto dendrite shafts and soma, not typically on spines.</li><li>Inhibitory interneurons play specific roles in shaping neuronal output:<ul><li>Martinotti cells: target distal dendrites.</li><li>Basket cells: target soma and proximal dendrites.</li><li>Chandelier cells: target the axon initial segment (AIS).</li><li>Some inhibitory cells also target other inhibitory interneurons.</li></ul></li><li>Synapses onto axon terminals can modulate transmitter release probability (presynaptic modulation) via modulatory transmitter actions.</li><li>Overall, neurons receive excitatory, inhibitory, and modulatory inputs at distinct subcellular compartments, which together determine output firing patterns. Synapses are highly packed, at densities of around$ 10^9 $synapses per cubic micrometer.</li></ul>3.27 Electrical synapses (Box 3-5)<ul><li>Electrical synapses (gap junctions) provide fast, bidirectional transmission via connexins (mammals) or innexins (invertebrates) and possibly pannexins.</li><li>Gap junctions are direct cytoplasmic connections between cells, formed by a pair of hemi-channels (connexons), each consisting of six connexin subunits. The distance between cell membranes reduces from$ \sim20 $nm to$ \sim4 $nm at gap junctions.</li><li>They allow passage of ions and small molecules; junctions form pores$ \sim1.4 $nm in diameter and enable dye-coupling experiments, demonstrating direct molecular transfer (e.g., dye filling in brainstem neurons).</li><li>Electrical synapses are common in circuits requiring rapid transmission and synchronization (e.g., retina, escape circuits).</li><li>In cortex, electrical synapses exist mainly between inhibitory interneurons (e.g., FS and LTS types) and can synchronize activity across networks; connectivity is cell-type specific.</li><li>Properties of Electrical Synapses:<ul><li>Graded Transmission: Response scales with presynaptic response; not an all-or-none signal like action potentials.</li><li>Fast Transmission: Lack synaptic delay, allowing very rapid signaling.</li><li>Bidirectional: Current can flow in both directions between coupled cells.</li><li>Non-inhibitory/Excitatory: Do not intrinsically hyperpolarize or depolarize in the same way chemical synapses do; primarily serve to pass charge directly.</li></ul></li><li>Advantages: Speed, graded response, bidirectionality.</li><li>Disadvantages: No all-or-none response, signal amplitude degrades over distance/time.</li></ul>3.28 Neuromuscular Junction Disorders<ul><li>Myasthenia Gravis:<ul><li>An autoimmune disease targeting nicotinic AChRs at the NMJ, causing grave muscle weakness and fatigability (often affecting small muscles like those of the eyes and facial expression).</li><li>Antibodies impair neuromuscular transmission by leading to receptor internalization, blocking receptor function, and causing complement-mediated membrane damage.</li><li>Diagnostically, the Tensilon test (administering edrophonium, a rapid-acting anticholinesterase) temporarily increases ACh in the synaptic cleft, which often improves muscle strength and can confirm the diagnosis.</li></ul></li><li>Lambert-Eaton Syndrome:<ul><li>A rare autoimmune disorder characterized by antibodies against presynaptic voltage-gated Ca2+ channels at the NMJ.</li><li>This leads to decreased neurotransmitter release from the presynaptic terminal.</li><li>Symptoms clinically resemble Myasthenia Gravis but can be differentiated by a negative Tensilon test.</li><li>Treatment often involves methods to remove autoantibodies, such as plasmapheresis.</li></ul></li></ul>Box 3-2: From toxins to medicines (highlights)<ul><li>Toxins target various steps of neurotransmission (Na+ channels by tetrodotoxin, Ca2+ channels by$ \omega $-conotoxin, SNAREs by botulinum and tetanus toxins).</li><li>Toxins have been essential tools and have inspired medical uses (e.g., Botox for muscle relaxation).</li></ul>Box 3-3: G proteins as molecular switches (overview)<ul><li>G proteins toggle between GDP-bound (inactive) and GTP-bound (active) states; regulators include GEFs (Guanine Nucleotide Exchange Factors), which promote GDP$ \to $GTP exchange (activating the G protein), and GAPs (GTPase-Activating Proteins), which increase GTPase activity to hydrolyze GTP to GDP (inactivating the G protein).</li><li>The GTPase cycle is central to GPCR signaling and cross-talk with other signaling pathways (Ras, Rab, Rho families).</li></ul>Box 3-4: Signal transduction and receptor tyrosine kinase (RTK) signaling<ul><li>Receptors can be GPCRs or RTKs; RTKs (e.g., Trk neurotrophin receptors) are activated by dimerization and transphosphorylation of tyrosine residues, providing docking sites for adaptor proteins (SH2/PTB domains).</li><li>Neurotrophin signaling through Trk receptors activates Ras-MAP kinase cascades (Ras$ \to $Raf$ \to $Mek$ \to$$ Erk) and transcriptional responses that promote neuronal survival and differentiation.
- CREB phosphorylation and opioid/growth signaling can cross-talk and regulate gene expression.
Box 3-5: Electrical synapses (expanded)
- Detailed experimental evidence in mammalian cortex shows specific connectivity patterns among inhibitory neuron types; FS, LTS, and RS neurons form diverse electrical synapses with directionality and specificity.
3.29 Summary (condensed)
- Neurons communicate via electrical and chemical synapses. Chemical synapses offer incredible flexibility in synaptic strength, a crucial advantage for processes like thinking and memory, as their efficacy can be adjusted through various mechanisms including changes in presynaptic Ca2+ influx, neurotransmitter release in response to Ca2+, neurotransmitter clearance rates, and the number of postsynaptic receptors.
- Electrical synapses are fast, bidirectional, and often used for synchronization; chemical synapses are unidirectional and rely on transmitter release.
- Release at presynaptic terminals is mediated by vesicle fusion; action potentials trigger Ca2+ influx via voltage-gated Ca2+ channels at the active zone, enabling synaptic vesicle fusion through a SNARE/SM complex.
- Excess neurotransmitters are degraded or recycled; vesicles are refilled via transporters and proton gradients.
- A common set of neurotransmitters operate across the nervous system; transmitter identity, receptor type, and subunit composition shape postsynaptic responses.
- Receptors are either ionotropic (direct ion flow) or metabotropic (GPCR-mediated signaling). Ionotropic receptors drive rapid changes; metabotropic receptors modulate excitability and signaling over longer timescales.
- GPCR signaling features amplification and termination, and includes both pre- and postsynaptic modulation.
- Postsynaptic responses are integrated by dendrites and soma, with spatial and temporal summation shaping the neuron's output; dendritic spikes and back-propagating action potentials modulate synaptic plasticity.
- Activity-dependent gene expression links synaptic activity to long-term neuronal changes via IEGs and transcription factors such as CREB and Fos; epigenetic modifications can further regulate gene expression.
- Proper organization of pre- and postsynaptic structures (active zones, PSD, adhesion molecules) ensures efficient and precise signaling.
- Electrical synapses provide rapid, bidirectional flow and synchronization in specific circuits; their presence is highly cell-type specific.