Exam Two - Molecular Biology of Neuron

Gap junction hemi-channels

• M1-4 transmembrane domains → form connexin

• 6 connexins need to come together to form a hemi-channel

• Hemichannels at pre- and post-synaptic membrane = align adjacent to each other. ⇒ form gap junction

Gap junction characteristics

• Have large diameter = allow non-selective ion passage. 

• Symmetrical synapse → cannot define pre- and post-synaptic membrane (no PSD)

Comparisons between channels & ion selectivity

• Voltage-gated cation channels: tetramers (4 subunits) = small, selective.

• Transmitter-gated ion channels (ligand-binding): 5 protein subunits forming central pore.

• Gap junctions: 6 connexins → greater diameter = even less selectivity

Gap-junction Transmission: Electrical synapse

• Transmission can be bi-directional; permit direct passive flow. 

• Transmission is fast ⇒ 0.2 ms in electrical vs. 2 ms in chemical

• Electrical activity is synchronized

• Electrical synapses do not have gain: induced response is always smaller than original signal in the source neuron. 

• Response always the same sign as source (excitation cannot produce inhibiting response)

Evidence for electrical synapse

• Action potential in crayfish: first discovered in crayfish nervous system. 

  • Found in neural systems that require fastest possible response (e.g. defensive reflexes) 

  • Synchronization of electrical activity among population of neurons like brainstem neurons (e.g. generating rhythmic electrical activity for breathing) 

  • Measured membrane potential in pre- and post-synaptic neuron: 

    • Action potential & EPSP response in post-synaptic neuron shows minimal delay (<0.1 ms) 

• Action potential in hippocampal interneurons = immediate post-synaptic response

Chemical synaptic transmission: cycle

• Vesicle docks: 

  • Cytoskeleton guides vesicle to pre-synaptic membrane (directly adjacent to PSD & receptors). 

• SNARE complexes form to pull membranes together

  • SNARE proteins: SNARE complex proteins all contain coil domains called “SNARE motif” → Tight binding

    • Synaptobrevin (on synaptic vesicle membrane)

    • On pre-synaptic membrane: Syntaxin & SNAP-25

• Entering Ca2+ binds synaptotagmin 

• Ca2+-bound synaptotagmin catalyzes membrane fusion

  • With calcium binding: Synaptotagmin inserts into phospholipid = interact with membrane = release NTs as vesicle fuses 

• Removal of Ca2+ from the cleft by astrocytes; recycling at level of pre-synaptic button.

EXPERIMENT: Otto Loewi’s evidence for chemical neurotransmission

• Hypothesized that chemical NT must be released for communication of nerve impulses.

• Isolated frog hearts: 1 incubated in beaker w/ fluid (continue to beat) 

  • Stimulate the vagus nerve = heartbeat of heart 1 slows down. 

  • If NT is released = 

    • Attachment of heart 1 to heart 2 → chemical NT released into fluid of beaker 1 that is connected to beaker 2 by a tube = affects heart 2 by slowing down its heart-beat.

  • Evidence of a secretory ligand that then inhibits heart 2. 

• Neurotransmitter: Acetylcholine (parasympathetic innervation) 

Small vs. large-molecular transmitters

• Small-molecule neurotransmitters: contained in small clear-cored vesicles (40-60 nm in diameter) 

  • Acetylcholine

  • Purines (ATP, GTP, adenosine) 

  • Amino acids (excitatory: glutamate, aspartate; inhibitory: GABA, and glycine) 

  • Biogenic amines (catecholamines [dopamine], indoleamine [serotonin], imidazoleamine) 

• Neuropeptides: more than 100 peptides, usually 3-30 amino acids long. 

  • Contained in large dense-cored vesicles (90-250 nm in diameter)

Vesicle synthesis: small-molecule transmitters/vesicles

• Synthesis of enzymes occurs in the cell body

• Slow axonal transport of enzymes into terminal

• Precursors (broken down after NTs have been released) are transported back into terminal from cleft. 

• Synthesis & packaging of NT at pre-synaptic button (axon terminal)

Vesicle synthesis: large dense-core vesicles

• Large dense-core vesicles budded off from Golgi & synthesis of NT precursors and enzymes at the cell body. 

• Transport down microtubule tracks (fast) ⇒ 400 mm/day

• Enzymes modify precursors to produce peptide NTs

EXPERIMENT: Difference in rate of release = differences in spatial arrangement of vesicles

• Low-frequency stimulation: 

  • Localized increase in Ca2+ ⇒ causes preferential release of small clear-core vesicles to be released (they are closer to the membrane)

    • Therefore, small-molecule NTs “act” faster. 

• High-frequency stimulation = more diffuse increase in Ca2+ concentration 

  • Both types of transmitters (clear-core & dense-core) are released 

  • Therefore, release of vesicles depends on concentration of Ca2+ 

EXPERIMENT: Testing calcium dependence of vesicle release

• Voltage-clamp & set presynaptic membrane potential to -25 mV 

  • Observed inward presynaptic calcium current

  • Post-synaptic membrane response observed. 

• Block Ca2+ channel ⇒ reduce presynaptic calcium current → no postsynaptic membrane response.

Synaptic vesicle cycle

1. Recycling: Vesicles formed through clathrin-dependent endocytosis. 

2. Empty vesicles merge into early endosome 

3. Undergo NT uptake 

4. Trafficked onto the active zone (zone of pre-synaptic membrane w/ vesicles aligned/docked & primed by ATP) 

5. Primed vesicles will be the first to fuse with the membrane. 

   1. Cav is located adjacent to primed vesicles. 

Synaptic vesicle cycle: Protein-protein interactions

Protein-protein interactions are essential for synaptic vesicle cycle to occur. 

• Can make antibodies to label these proteins. 

  • SNARE Protein: syntaxin, snap-25, synaptobrevin ⇒ synaptic vesicle proteins

  • Synaptotagmins on the membrane

  • Piccolo & Bassoon: active zone proteins 

• MINT proteins: bind to synaptic vesicle proteins (neurotransmission) & interact w/ Alzheimer’s disease proteins. 

Synaptic vesicle pools

• Readily-releasable pool 

  • 1-2% docked at active zone → released <1 second with fast recycling

• Recycling pool

  • 10-20% scattered 

• Reserve pool

  • 80-90% scattered → 10s of seconds-minutes; slow recycling (minutes) 

  • Bulk of vesicle cluster.

EXPERIMENT: Order of synaptic vesicle release

• Can tag synaptic vesicles w/ fluorescent tag ⇒ detect fluorescence changes in extracellular media. 

  • Over time, if you continuously excite pre-synaptic neuron ⇒ observe sequence: RRP released first, then followed by the others. ⇒ will eventually depleting the pool

Katz’s model system: Neuromuscular Junction

Model system: frog’s neuromuscular junction model: neuron’s synaptic button ⇒ release ACh ⇒ bind to ACh receptors on post-synaptic muscle membrane = contraction. 

• Large junction = easy to visualize 

Stimulate the axon w/ current & record post-synaptic membrane potential of muscle cell. 

• End-plate potential (EPP) ⇒ transient depolarization of post-synaptic membrane potential in response to action potential

  • If large enough → can trigger action potential. 

EXPERIMENT: Katz’s seminal study - spontaneous membrane potential changes

Even without stimulation, there are spontaneous changes in post-synaptic membrane potential ⇒ Miniature end plate potentials (MEPPS)

• Experiment 1: Pre-synaptic modification ⇒ Removed calcium (responsible for NT release) ⇒ no more MEPPs in muscle cell

• Experiment 2: Post-synaptic modification ⇒ Curare (ACh receptor inhibitory) ⇒ no more MEPPs in muscle cell

EXPERIMENT: Katz’s quantal mechanism of NT release

Hypothesis: MEPPs are spontaneous events that respond to fusion of synaptic vesicles (release of NTs) at rest (w/o action potential) ⇒ Is it caused by one molecule of ACh or by many molecules? 

• Mathematician: Proposed that 10,000 ACh/vesicle = quanta

• Experiment 3: Vary the concentration of acetylcholine (1 vs. 10000 molecules) 

  • Finding:

    • Each vesicle = one quantum of transmitter amounting to several thousand molecules. 

    • Each quantum produces post-synaptic potential of fixed size (quantal amplitude) 

    • Neuron’s response is the sum of individual quanta 

      • Action potential accelerates and synchronizes release of quanta. 

EXPERIMENT: Quanta and synaptic vesicle fusion

Quanta result from fusion of individual synaptic vesicles with the plasma membrane. 

• Stimulate the pre-synaptic membrane⇒ can see vesicles fusing with plasma membrane. 

• Positive linear correlation between number of quanta released into synapse & number of vesicles fusing.

Modes of NT release: Evoked

• Evoked synchronous release: initiated within milliseconds after action potential

• Evoked asynchronous release: sets in w/ delay after action potential. 

  • Normally negligible: hidden by synchronous release or synaptotagmin suppresses async release.

    • Experiment: Knockout of synaptotagmin-1 (essential for evoked synchronous release) ⇒ can still visualize asynchronous release; therefore, synaptotagmin-1 is not essential for asynchronous release.

  • Purpose: May shape neural function at circuit level; may maintain synaptic vesicles.

Modes of NT release: Spontaneous

Spontaneous “mini” release: exocytosis of a single vesicle that is independent of action potentials but dependent on Ca2+ 

• Minis (mEPSC) are also observed in WT neurons 

• Fusion machinery (proteins) are also unique to evoked synchronous release.

Synapsins

• Maintaining reserve pool

• Mobilization of synaptic vesicles: 

  • Synapsin is the “highest” protein in the brain that can be phosphorylated → regulated by this post-translational modification. 

Synapsin phosphorylation

• Dephosphorylated synapsin I (inactive): bound to synaptic vesicles & binds them to the nerve terminal cytoskeleton (actin) ⇒ holds at reserve pool

• Phosphorylated synapsin I ⇒ release from vesicles = free vesicles from cytoskeleton & make them available for exocytosis. 

  • Phosphorylation can occur through secondary cascades

    • G-protein activated in response to NT binding → activates adenylyl-cyclase (AC = secondary messenger) → cAMP kinase → phosphorylation of synapsin I

Experiment: Synapsin triple knockout mice

Must knockout all three synapsins (6 alleles) to observe clear phenotype, as they compensate for each other.

• Finding: 

  • Viable and fertile 

  • However: seizures and behavioral deficits. 

Experiment: Electron micrograph of synaptic vesicles in synapsin KO

Electron micrograph of pre-synaptic excitatory synapse (asymmetric: have pre-synaptic vesicles & thick PSD; inhibitory synapse has a finer PSD)

• Observed mean number of synaptic vesicles at increasing distance from active zone: 100, 200, 300, 400, 500 nm

  • KO: Further away = reduction in # of SV ⇒ likely affecting the reserve pool. 

  • Not much changes in docked vesicles compared to WT.

Experiment: Reserve pool depletion in synapsin KO

• Experimenter can keep stimulating the neuron ⇒ triple KO will deplete in reserve faster

• Plotted # of stimuli & normalized EPSC current to see where depletion occurs. ⇒ TKO curve drops faster at around 50 stimuli.

  • Depression time constant ⇒ faster depression in triple KO.

RIM Proteins

Docking of synaptic vesicles at pre-synaptic membrane 

• Are adapter protein that links synaptic vesicles to Cav. 

  • Rab3: connection point between RIM and synaptic vesicle.

  • RIM-BP and Munc13 are also involved in complex. 

EXPERIMENT: Conditional double-knockout RIM

• Conditional double-knockout (RIM1&2): number of vesicles docked at active zone is significantly reduced. 

  • However, vesicles per button have not changed. 

• Experiment:

  • Small EPSC & IPSC from cDKO 

  • Also examines mEPSC: frequency is reduced.

Syntaxin

Part of SNARE complex

• N-terminus: HA,B,C domains → SNARE motif → trans-membrane region (TMR) 

• Has open and closed conformation:

  • Auto-inhibited: folds upon itself (HA-C folds on SNARE motif).

  • Open conformation: can bind to SNAP-25 & Synaptobrevin

  • Responsible protein: Munc18 ⇒ binds well to closed Syntaxin & must dissociate for syntaxin to open. 

EXPERIMENT: Munc-18 binding

Method: Western blot

• Syntaxin is tagged with GST (bait) that sticks to beads

  • Make mutated versions of various amino acids of syntaxin (e.g. Leucine165 & E166).

• Homogenized rat brain tissue: membrane-bound proteins (Munc18, SNAP-25, synaptobrevin) dissolved in mild detergent and can bind the bait.

• Beads incubated w/ lysate & washed = release bait & binding partners that can be separated by size on gel

  • Western blot: Identifies the protein pulled-down.

• Findings: 

  • Munc-18 binding is lost when we mutated at 165 & 166

  • Where Munc-18 could not bind, Snap-25 & synaptobrevin could 

  • Negative controls: Synatogyrin & Rab3A shows no band = no interaction w/ Munc-18

EXPERIMENT: Knock-in mutation of Leucine165 & E166

Knock-in mutation in vivo ⇒ mutated Leucine165 & E166 into Alanine ⇒ mice demonstrates behavioral deficits.

Mechanisms of Endocytosis

Clathrin-dependent endocytosis: 

1. Clathrin bind to one another⇒ triskelion structure ⇒ involved in invagination of the membrane. 

2. Dynamin ring pinches off the membrane 

3. Vesicle (coated by clathrin) are translocated by actin filaments. 

4. Clathrin is removed by Hsc-70/auxilin 

Experiment: local vesicle recycling in presynaptic terminals

Treat neurons w/ horseradish peroxidase in extracellular medium. 

• HRP can be taken up by synaptic vesicles → wash away extracellular HRP → wait 5 minutes → follow where they go. 

  • Process: total 1 minute

    • Fusion (1 ms) → endocytosis & budding (10-20 seconds) → endosome → budding from endosome → docking → priming → exocytosis. 

Alternative method: FM1-43

• Voltage-sensitive dyes

  • Has a lipophilic tail & carries a fluorophore and positive charge. 

  • Treated with HRP in extracellular medium = follow fluorescent protein ⇒ visualize both endocytosis & fusion events.

Neuropathologies: Synaptic transmission

• Familial infantile myasthenia: reduces vesicle size

• Congenital myasthenic syndromes: impaired vesicle recycling. 

• LEMS (antibody) can cross BBB & attack presynaptic Ca2+ channels in autoimmune disorders ⇒ Synaptic transmission won’t occur

Experiment Method to Study Synaptic Transmission: Toxins

• Toxins that trigger vesicle fusion in pre-synaptic membranes: 

  • Neurexins: initially detected as receptors for alpha-latrotoxin (component of black widow spider venom)

    • Causes MASSIVE NT release. 

    • Possible hypothesis: Cell-adhesion molecules ⇒ perhaps interfere w/ pre-synaptic membrane = Ca2+ influx independent of Cav. 

• Toxins that affect SNARE proteins: 

  • Botulinum (BTX ⇒ e.g. botox): inhibits neurotransmitter release 

  • Tetanus toxin: inhibits release of inhibitory transmitters ⇒ hyper-excitability

Synaptic plasticity

Short-term: Few minutes or less => Largely based on pre-synaptic event: changes in amount of NT released & synaptic vesicle pools. 

• Alterations in Ca2+ signaling = important

Long-term (>30 mins): Largely based on Ca2+ and other intracellular secondary messenger pathways. → takes place at the postsynaptic level.

Short-term Facilitation

Facilitation: enhances NT release by persistent action of Ca2+ ions

• When 2(+) APs invade presynaptic terminal within 10 ms of each other → greater amplitude of post-synaptic membrane potential. 

  • Due to prolonged elevation of pre-synaptic Ca2+ levels = more release of vesicles.

• Facilitation decays w/ longer the interval between stimuli ⇒ therefore, the pairs must arrive CLOSE together.

Molecular mechanisms of Short-term facilitation

• Serotonin released from facilitator interneuron (modulatory) → bind to G-coupled receptor that activates G-alpha-s (Gαs)

• Gαs → activates adenylyl cyclase → converts ATP into cAMP 

• cAMP binds regulatory subunit of PKA → release catalytic subunit that phosphorylates K+ channels = close

• Less K+ = AP repolarizes more slowly = Ca2+ channels open longer (increase Ca2+) 

  • More Ca2+ = more Glutamate vesicles released = larger EPSP

Short-term Depression & Experiment

Depression: decreases amount of NT due to depletion of RR-vesicles. 

• Experiment: consistent stimulation over 5s ⇒ big drop in post-synaptic response. 

  • Dependent on Ca2+: 

    • Lower concentration of Ca2+ {intermediate, 0.1 normal) during stimulation: does not lead to depression → leads to augmentation. 

      • Lowering Ca2+ availability = decreased efficiency of fusion. 

  • More relative amount of NT release = more relative amount of depression.

LTP Mechanisms

• CAMKII (involves calmodulin) & insertion of AMPA receptors to modify synapse excitability. 

  • AMPA/NMDA: AMPA responds first to GLU ⇒ leads to Na+ influx → removes Mg2+ plug of NMDA, so GLU binding can allow Ca2+ to come in → Ca2+ binds calmodulin & activates CAMKII → phosphorylates downstream substrates (effector proteins) ⇒ inserts additional AMPA receptors. 

• Morphology of dendritic spines (mushrooms)

Long-term plasticity model: Hippocampus

Unidirectional information movement

• Entorrhinal cortex (EC) sents axons along perforant-path → synapse on dentate gyrus → sends axons to synapse w/ Ca3 region (pyramidal neuron) 

• Ca3 synapse on Ca1 neuron → back into EC

• Slice physiology matters in study of LTP

Experiment: Hippocampus induction of LTP

Stimulate Ca3 and record EPSP in Ca1 neuron 

• High-frequency stimulation (100 Hz) for long period of time: long-term potentiation ⇒ Ca1 recording enhanced for long period of time (hours) 

• Low-frequency stimulation (1 Hz): lead to long-term depression ⇒ EPSP decreased over time

LTP characteristics

• Input specific: to a specific synapse

  • Pathway 1: active ⇒ synapse strengthened 

  • Pathway 2: inactive ⇒ synapse not strengthened 

• Associative: 

  • Pathway 1 has strong stimulation & Pathway 2 has weak stimulation ⇒ however, if they fire at the same time = can strengthen both synapses. 

Molecular Mechanisms of Long-term Facilitation

Repeated or strong serotonin exposure

• cAMP stays high so catalytic PKA move into nucleus → phosphorylates CREB 

• CREB (transcription factor) turns on gene expression

• Output: New gene expression and structural change

  • C/EBP transcription factors continues to “switch on” genes.

  • Ubiquitin hydrolase degrades PKA regulatory subunit → PKA stays active even w/o cAMP 

  • Proteins for synaptic growth build new synaptic connections between sensory and motor neurons.

Long-term depression

Reduction of receptors through endocytosis → decrease postsynaptic response.

LTD: Hippocampus

1 Hz stimulation

• Internalization of AMPA receptors ⇒ Ca2+-dependent. 

  • Glutamate activates AMPA = Na+ influx & NMDA = Calcium influx ⇒ Calcium activates protein phosphatases ⇒ dephosphorylate substrates ⇒ internalization of AMPA receptors.

LTD: Cerebellum

• AMPA receptors endocytosed ⇒ BUT DIFFERENT PROCESS

  • Glutamate activates metabotropic glutamate receptors (GPCR) → activates G-alpha-q → activates phospholipase C & DAG ⇒ increase Ca2+ 

    •  Ca2+ → activates PKC → phosphorylates substrate proteins → long-term depression through internalization of AMPA receptors

• Associative activation: Presynaptic terminal of parallel fiber & climbing fiber = synapses must be activated at the same time for LTD to occur. 

  • Parallel fiber = DAG from mGluR; Climbing fiber = large Ca2+ influx ⇒ strong activation of PKC.

Kandel’s Model System: Siphon (Aplysia) - Behavioral

• Non-noxious model system: Gill contracts every time the siphon is “tickled”

  • Over time, gill response decreases significantly (habituation) 

• Noxious stimuli (inflict pain): shock the tail = muscle contracts. 

  • Touch siphon + shock = siphon becomes more sensitive to pain (sensitization)

Experiment:

• With single tail shock = response spikes compared to no shock but eventually goes away → short-term sensitization

• 4 trains of tail shocks or 4 trains/day for 4 days ⇒ amplified reflex lasts longer

  • Stimulus pattern determines whether effect is long/short-term.

Kandel’s Model System: Siphon (Aplysia) - Molecular system

• Siphon skin’s sensory neuron → synapses to motor neuron → causes gill contraction when it fires (excitation at neuromuscular junction) 

  • Interneuron connects to motor neuron ⇒ over time, inhibits motor neuron response to non-noxious stimulus.

  • EPSP decreases over time. 

• However, stimulate the tail → activate modulatory interneuron (serotonin) → activates synapse between motor neuron & gill = causes firing. 

  • Restores EPSP in motor neuron (4th panel)

Endocannabinoids

Plant-derived: THC 

Endogeneous: 

• Anandamide (AEA) 

• 2-AG 

Affect neural functions: euphoria, appetite stimulation, sedation, etc. 

G-protein coupled receptors: CB1 (pre-synaptic terminals or axons), CB2

CB1: Post-synaptic (making of messengers)

• Glutamate binds to mGluRs & ionotropic receptors post-synaptically → activates G-alpha-q  (Gaq)

• Gaq activated = PLC-beta is activated = cuts PIP2 = DAG and IP3 → increase in Ca2+ 

• Ca2+ & DAG stimulate enzymes converting membrane precursor into AEA or 2-AG = diffuse into cleft. 

  • Recycling: Transporter takes endocannabinoids back into post-synaptic cell & enzyme breakdown into precursors 

CB1: Pre-synaptic (localization & effect)

• AEA or 2AG binds to CB1 pre-synaptically → activates the G-alpha-i pathway → inhibits adenylyl cyclase (AC) to decrease ATP, cAMP, PKA 

• Additionally, negative feedback regulation to NT release: Beta-gamma subunits can activate Kv and inhibit Cav ⇒ inhibits NT release

Experiment: Depolarization-induced (glutamate) suppression of inhibition (inhibit GABA-ergic neurons)

• Requires increase in postsynaptic calcium ⇒ associated w/ decrease in IPSC frequency and not amplitude; GABA has no effects. 

  • Increase in Ca2+ after innervation by glutaminergic afferent ⇒ production of 2AG ⇒ binds to CB1 in GABA-ergic afferent ⇒ inhibition of NT release. 

• Experiment: 

  • Depolarizing pulse to post-synaptic neuron → transient decrease in IPSC response 

  • Treat with AM281 (antagonist of CB1) → maintained IPSC 

Experiment: LTP & Cannabinoids

LTP inhibited by CB1 receptor blockade 

• 100 Hz stimulation (Theta-burst stimulation: TBS) = LTP observed 

  • Can also apply reagents that enhance LTP (chem-LTP): adenylyl cyclase activator (e.g. forskolin) and phosphodiesterase inhibitor in presence of low Mg. 

• Both LTPs can be inhibited by CB1 competitive receptor NESS-0327 OR CB1 receptor inverse agonist

Homeostatic Plasticity & Experiment w/ Toxins

• Negative feedback mechanisms that neurons use to offset excessive excitation (excitotoxicity due to excess Ca2+) or inhibition by adjusting synaptic strength. 

• Experiment: culture neurons

  • Treat with TTX for long period of time (hours): Blocks voltage-gated Na+ channels = reduces network activity. 

    • Compensatory mechanism: insert more receptors at the postsynaptic site. 

  • Treat with Bicuculline: Blocks ionotropic GABA-A receptors (inhibitory)

    • Compensatory mechanism: endocytosis of receptors.

Arc protein

Mediates homeostatic synaptic scaling of AMPA receptors 

• Regulated by neuronal activity in primary culture

  • Bicuculline = increased activation of neurons = increase Arc = decrease in surface AMPA receptors.

  • TTX = decreased network activity = decreased Arc expression.

• Highly expressed post-synaptically. 

Homeostatic Plasticity: Glial cells

Blocking network activity w/ TTX → decrease Glutamate from neurons → glia sense activity loss and release TNF-alpha (soluble factor) = scaling up excitatory synapses. 

Experiment: TNF-a

TNF-a mediates synaptic scaling during activity blockade. 

• Treat with TNF-alpha & chronic treatment w/ TTX = increase in AMPA-receptors surface expression. 

• mEPSCs: 

  • Chronic activity blockade = increase in mEPSC amplitude due to AMPA-r increase .

  • Conditioned medium from TTX-treated cultures applied to untreated neurons = causes increase in mEPSC amplitude. 

    • To confirm that TNF-alpha is contained in the conditioned media (CM) ⇒ treat CM w/ sTNFR.

      • sTNFR bind TNF-alpha ⇒ no TNF-receptor activation ⇒ no increase in mEPSCs amplitude. 

Case study: H.M.

• Seizures from bicycle accident in youth ⇒ bilateral lesion of medial temporal lobe, MTL (contains the hippocampus = important for learning & memory).

• Severe anterograde amnesia: unable to establish new memories (consolidation of “recent memory”) 

• Temporally graded retrograde amnesia: memories created prior to the event are lost. 

• In-tact:

  • Working memory: temporary storage & manipulation of information, operates over a few seconds. 

  • Procedural memory.

  • Preserved older memories (remote memory ⇒ stored at level of cerebral cortex)

• Therefore, hippocampus functions as temporary storage for new information, but LTM/permanent storage (consolidation) depends on distributed cortical networks. 

Spatial Learning & Hippocampus

Paradigm - Morris Water Maze: mouse placed in opaque pool at random start point → locate platform. ⇒ latency SHOULD decrease over time as the animal learns spatial cues.

• Hippocampal lesion: mouse can no longer build/use a spatial map of the maze & surrounding environments. → path not direct to platform like controls.

Synthesis of place & grid cells in rodent brain

Grid cells in EC recognize head direction of animal → GPS system w/ place cells in hippocampus (form synapses) → causes firing of place cells.

• Grid cells ⇒ how far & what direction it has moved. 

• Place cells ⇒ where it is.

Experiment: O-Keefe’s Place Cells

O’Keefe’s Experiment: Hippocampus as spatial reference map

• Electrodes placed on rat’s hippocampus to record single neuron firing (A, B, C, etc.) as rat moves around rectangular box. Curtain hides external cues. 

• Finding: 

  • Neuron fires strongly at A, and less or not at all at other positions.

    • Therefore, subpopulation of neurons sensitive to spatial position ⇒ activated upon specific directionality. 

  • O’Keefe discovered “place cells” located in hippocampus

Experiment: Moser & Moser “Grid Cells”

• Identified nerve cells in EC that were activated & form hexagonal grids that react to unique 

• Form coordinate system = allows for spatial navigation 

Learning & consolidation: synaptic level

Very fast; morphological changes = necessary for initial stabilization of memories in hippocampal circuits (e.g. growth of new synaptic connections, restructuring of existing synaptic connections = e.g. spine morphology) 

• Synaptic activation

• Activation second messenger systems: e.g. PKA, CAMKII

• Transcription factors & protein synthesis ⇒ e.g. CREB

Learning & consolidation: system level

Slow: Gradual process of reorganization of brain regions that support memory. 

⇒ Over time, connections between hippocampus & cortical modules weaken as objects are stored. Circuit between cortical modules become strengthened to store remote memory (refer to figure.) 

• Sleep is important for memory consolidation over time ⇒ strengthens cortical consolidation. 

PFC and Remote Memory

• Recent memory: HPC strongly linked to cortical modules (hub); PFC weakly engaged ⇒ recall is hippocampus-dependent

• Remote memory: cortical modules + PFC strongly interconnected; hippocampus connections weak ⇒ recall is hippocampus-independent

  • PFC inhibits the hippocampus as the memory matures ⇒ memory maintained by the cortical network (systems consolidation)

Behavioral paradigm: contextual fear conditioning

Pavlovian condition where animal learns association between distinctive place [context] and aversive event [shock]. 

• Single training experience = freezing behavior even w/o electrical shock response in the future.

Experiment: Contextual fear memories & hippocampus

• Lesions to hippocampus either 1, 7, 14 or 28 days after training. 

  • 1 day: no longer freezes = has not “learned” behavior

  • 28 days: same freezing profile as control 

    • Long-term memory located in the cortex 

• Finding: HPC lesions produce temporally-graded retrograde amnesia for contextual fear memories in rats. 

Behavioral paradigm: 8-arm radial maze

W/ novel objects on some arms. 

• Animal will explore the arms & usually gravitate towards the novel object. 

• Test recent memory (5 days): animal can go back to the arm w/ the object. 

• Test for remote memory after long retention interval: 

  • Same context, same baited arm: animal can re-learn route. 

  • Different context, different baited arm = back to recent memory —> benefit of switching back & forth

Tracing activity in 8-arm maze paradigm

• Can inject radioactive 2-DG intravenously: 

  • Retention testing → brain removal & sectioning → autoradiographs. 

  • Finding: 

    • Hippocampal level: lighting up for recent memory 

    • Remote: anterior cingulate cortex (ACC) lights up; HPC no longer lights up. 

    • When switching back to recent memory w/ the same paradigm: HPC lights up again. 

• NOW: Can do C-Fos staining & immunostain for activity. 

Opsins

Light-gated ion channels

• Sensory photoreceptors in green algae controlling photoaxis (movement towards light) 

• Can be expressed onto neurons 

• Functions: 

  • Channelrhodopsin: blue light & allows Na+ ions to enter ⇒ spiking activity in neuron.

  • Halorhodopsin: responds to yellow light & allows Cl- ions to enter. ⇒ inhibition

Channelrhodopsin specificity: Plasmid design

• Contains ChR & fluorophore (see which cells are expressing ChR) 

• Also has promoter: can be specifically selected

  • Example: CAMKII promoter ⇒ only expressed in CAMKII+ excitatory neurons

  • Example: GAD65 promoter: only in inhibitory GABA-ergic neurons.

• WPRE, RRE ⇒ viral components 

  • Neurons are post-mitotic = difficult to express DNA readily. HOWEVER, viruses can infect neurons well. 

    • Culture = lenti-virus

    • Rat: adenovirus as packaging systems.

Experiment: Characterization of ChR

• Hippocampal neurons labeled w/ GFP & co-expressed CAMKII. 

• Shining blue light = inward current response throughout the whole duration of the light being on. 

  • Increase in Hz of light ⇒ increased in frequency of AP responses. 

Rhodopsin: Halorhodopsin in hippocampal CA

• Inject viruses (eNpHR) into hippocampus at different Bregma sites (intersection between frontal & parietal bones of skull) 

  • Halorhodopsin expressed in CA region of hippocampus.

• Finding: 

  • Turning yellow light on through double cannula guide = no firing (decreased frequency) ⇒ creates temporary lesion 

Experiment: CA1 inhibition & contextual fear acquisition/retrieval

CA1 optogenetic inhibition blocks contextual fear acquisition & retrieval

• Mice placed in cage & pure tone is paired with foot shock = induce fear conditioning in controls. 

• In mice w/ halorhodopsin: 

  • Light on during testing (in same chamber) = no longer freezing 

  • Light off = mice w/ halorhodopsin can learn fear conditioning well. 

Experiment: Auditory-cue fear memory

• Design: 

  • Fear-conditioned mice placed in pyramid-shaped cage w. smooth floor (different set-up).

  • 2.9 kHz tone was sounded for 2.5 minutes. 

  • Freezing behavior was measured over 2.5 minutes

• No difference between control & eNpHR mice w/ light on or off

Experiment: eNpHR & amygdala

• Paradigm: Mice in open field 

  • Presence of virus w/ halorhodopsin = not much change compared to control ⇒ no motor deficits to hinder movement. 

• Finding: eNpHR expression in basal lateral amygdala = inhibit amygdala neurons = loss of contextual & auditory-cue freezing response (memory acquisition)

Anterior cingulate cortex & remote memory

Anterior cingulate cortex optogenetic inhibition disrupts remote (1 month) but not recent (1 day) fear memory recall. 

• Expression eNpHR in ACC in one day = freezing response normal 

• Expression of eNpHR in one month = loss of learning response. 

  • Decreased # of c-FOS-expressing cells. 

Acetylcholine cycle

1. Glucose -> pyruvate -> Acetyl CoA

2. Acetyl CoA -> acetylcholine by choline acetyltransferase. 

3. Vesicular ACh transporter = packages ACh into NTs. 

4. Once released into the cleft, binds to acetylcholine receptors in postsynaptic cells. 

5. Can be broken down in the synaptic cleft -> acetylcholinesterase breaks acetylcholine into acetate & choline

   1. Choline can be recycled by Na+/choline transporter to pre-synaptic cleft. 

   2. UNIQUE PROPERTY COMPARED TO GLU/GABA: bcz astrocytes are not primary function to break down ACh. 

      1. Some early drugs for Parkinson’s inhibit acetylcholinesterase = increase availability in synapse.

Receptors

1. Ligand-gated ion channels: fast, use concentration gradient. 

• Pentameric: ion pore = larger ⇒ ion selectivity decreases. 

2. G-protein-coupled receptors: Rely on G-protein subunits that activate downstream messengers (G-alpha-i/s/q) = modulate ion channels. 

• Lefkowitz & Kobilka (2012 Nobel Prize): 

  • ~200 functionally known receptors, ~600 functionally unassigned receptors (orphan receptors) 

    • Sensory (taste, smell) & hormone receptors 

  • G-protein receptors have unique pockets ⇒ can bind drugs (Account for 60% of all prescription drugs.) 

Structure of G-coupled proteins

7-transmembrane segments w/ C & N terminal

• Heterotrimeric: 3 distinct proteins

  • Alpha: binds GTP (active) and GDP (inactive) → Ga is an GTPase.

  • Beta-gamma: functional unit = governs particular channels. 

    • E.g. activation of Kv channels and inactivation Cav. 

  • Binding of GTP to Ga causes Ga-GTP to dissocitate from Beta-Gamma 

  • Hydrolysis of GTP → GDP = “turns off” Ga (allows By to rejoin and resets the cycle.

Toxins to G-protein

• Cholera toxin: activates G-proteins

• Pertussis toxin: inhibits G-protein

Summary of G-alphas: Ga (toxin sensitive)

• Gαs: CTX sensitive (activated by Cholera toxin) 

  • Adenylyl cyclase activation → increased PKA ⇒ implicated in facilitation

    • Read out: increased cAMP

• Gαi: Sensitive to PTX (inactivated by pertussis toxin)

  • Inhibits adenylyl cyclase → decrease in PKA activation → decrease cAMP

    • Seen in: presynaptic CB1-coupled G-protein. 

  • Activates K+ (efflux), inhibits Ca2+ ⇒ overall hyperpolarizing effect. 

  • Examples: GABAb, opioid, mGluR2-4.

Summary of G-alphas: Gaq

Gαq: 

• Activates phospholipase C (PLC) ⇒ Breaks down PIP2 into DAG 

• DAG (diacylglycerol) increases IP3 → increase Ca2+ 

  • Implicated in: LTD in cerebellum; post-synaptic mGluRs’ G-protein (endocannabinoids synthesis pathway)

• Increased Ca2+ = read-out

• Examples: mGluR1,5; muscarinic M1 & 3, etc.

Nicotinic acetylcholine receptors

Ligand-gated 

• 5-subunits (pentameric): can be homomeric or heteromeric

  • Only alpha subunits bind to ligand

  • Delta & gamma subunits highly expressed at neuromuscular junctions. 

  • Structure: Ligand binding pocket, Gate, Central pore 

Assembly of nicotinic acetylcholine receptor

• Each subunit: Large N-terminal + 4 transmembrane segments + C terminal. ⇒ must come together to form functional channel. 

• Heteromeric: increases diversity of nicotinic-ACh channel. 

  • Alpha-4-Beta-2 (a4₂b2₃) ⇒ 2 alpha-4, 3 beta-2 

• Subunit composition and stoichiometry can influence:

  • Receptor desensitization: e.g. a7 = lower ACh affinity, rapid desensitization (closes fast)

  • Ligand affinity: e.g. a4₂b2₃ has higher affinity to ACh & nicotine

  • Conductance

Conformational states of nicotinic acetylcholine receptors

• Fast activation: high-affinity binding of ligand = opens quickly 

• Closed: at rest or low-affinity for agonist

• Desensitized state: channel closed & unresponsive to ligand

nAChR Toxin: Alpha-bungarotoxin

Structure: alpha-sheets 

• Experiment: a-Bungarotoxin inhibits muscle nAChR expressed in Xenopus oocytes 

  • 10 nM - 50 nM of a-Bungarotoxin ⇒ significant depression of inward current (Na+ or Ca2+) 

    • Higher concentration (50 nM) = complete inhibition. 

Lynx-1 Protein

Endogenous; morphologically similar to alpha-bungarotoxin. → similar inhibitory effects.

• Lynx genes belong to ly-6/PLAUR superfamily ⇒ prototoxins: ancestors to non-venomous alpha-neurotoxins. 

  • Modulators in governing control points in nicotinic receptor signaling pathways. 

• In-vivo: 

  • Highly expressed in brain; In-situ hybridization: MC of olfactory bulb, CA3, layers 5&6 of cerebral cortex

Experiment: Lynx1 coexpression w/ nAChR

Lynx1 enhances desensitization when co-expressed w/ nicotinic receptors

• Apply ACh to a4b2 receptor → inward current & prolonged current

• Presence of ACh + lynx 1

  • Accelerated onset of desensitization

  • Slows recovery of desensitization = molecular brake.

  • Reduced agonist sensitivity

Experiment: Lynx1 knockout

• Paradigm: monocular deprivation (close one eye for several days) = high plasticity in young mammals, but restricted in adults.

• In younger mice: the closed eye sends little activity so visual cortex neurons shift their responses toward the open eye (take over closed eye’s cortical territory).

• Knock-out Lynx1 in 60-day-old adult = exhibited plasticity that matches 30-day-old juvenile WT mice (after 4 days of MD).

  • WT adult mice do not show this plasticity.

    • Usally, Lynx1 inhibits nAChRs, so less cholinergic signaling, so less plasticity

Homomeric a7 nACHRs & Schizophrenia

• Cholinergic neurons release ACh → binds to A7 in GABA-ergic interneurons = excite interneuron. 

• Interneuron synapses to pyramidal cell = inhibits the cell = diminishes Glu

• Schizophrenia patients have abnormalities in filtering sensory stimuli that underlie hallucinations/delusions. 

  • Some studies have shown that schizophrenia patients have the greatest rate & intensity of cigarette smoking. 

    • Nicotine (in cigs) = transiently improves their sensory inhibition. 

Schizophrenia patients & sensory stimuli filtering

Experiment: Cerebral evoked P50 potentials to repeated sounds (e.g. background sounds) over time are inhibited in controls but not in schizophrenia patients. 

• Normal: decrease in response because the pyramidal cell is normally inhibited by the excited interneuron. 

• Mutated A7: pyramidal cell activated

Myasthenia Gravis

Autoimmune disease ⇒ produces antibodies that bind to nAChR ⇒ unable to bind ligands

• Results: profound weakness in muscles, diplopia, drooping of corners of mouth/eye (sorrowful appearance)  

• Plasmapheresis can help alleviate symptoms. 

Muscarinic ACh Receptors

GPCRs:

• Types: M1-5

  • M1, M3, M5: G-alpha-q ⇒ stimulatory = increases in Ca2+

    • Predominantly post-synaptic ⇒ always excitatory

      • Increase PLC → increases Ca2+ levels 

    • Can co-localize with glutamate AMPAR & NMDAR

      • Can enhance NMDA receptor activity: cross-signaling between GPCR & other ion channels

  • M2, M4: G-alpha-i ⇒ inhibitory = decreases in Ca2+ 

EXPERIMENT: M1,3,5 & NMDA Receptor activity

Can enhance NMDA receptor activity: cross-signaling between GPCR & other ion channels

• Experiment: Activation of M1 muscarinic receptor potentiates NMDA receptor currents in hippocampal cells. 

  • Drug: carbachol (CCH) = cholinergic agonist that activates muscarinic acetylcholine receptor. 

    • Stimulation of M1 receptor = potentiation effect in NMDA 

  • Treatment with atropine: nonspecific mAChR inhibitor to M1,3,5 ⇒ inhibits potentiated response. 

  • Treatment with M1-selective antagonist: decrease in % potentiation ⇒ not seen for M4-selective antagonist. 

M2, M4: Localization

Presynaptic: negative feedback ⇒ “stop” releasing NTs. 

• At cholinergic terminals: 

  • M2 mAChRs = autoreceptors that suppress ACh release & inhibit cholinergic NT at select CNS synapses. 

• At non-cholinergic neurons: 

  • M2 mAChRs are heteroreceptors: control release of other NTs (e.g. dopamine) 

• Experiment: 

  • Field EPSP & LTP effects (over 60 minutes)

    • Presence of CCH 

      • WT: good potentiation response

      • M2 KO: deficits in LTP response. 

Ionotropic glutamate receptors

Tetrameric (4 subunits), non-selective cation channels.

• NMDA receptors: Heteromers → at least 5 different forms (NR1 + NR2A-D come together)

• Non NMDA

  • AMPA: GluR1-4 

  • Kainate: low affinity or high affinity 

• Delta: orphan receptors

  • Do not exhibit agonist-induced ion channel currents; unresponsive to glutamate

  • May play a role in synaptogenesis