Dynamic Synapse

Spinogenesis definition

formation of new dendritic spines, excitatory neurons, glutamate neurons

Synaptogenesis definition

formation of functional synapses between neurons

What are dendritic spines? + purpose

Dendritic spines = tiny, dynamic protrusions from neuron dendrites that serve as the primary site for excitatory synaptic input in the brain

(there to ensure cell receives only relevant info) – threshold for passing on info likely to be different at different synapses

How do dendritic spines work?

è Axons speak to dendrites that receive the info + bring it to the cell body

è Cell body computes + decides on sending this info down or not

è If sent down: in form of AP to presynaptic terminal

è Goes to cortical neurons, found in frontal cortex of a mouse

Why have dendritic spines? (2)

1.    Increasing SA + synaptic connections

a.    Neuron can connect with more potential partners

2.    Able to compartmentalize electrical + biochemical signals from the cell

a.    Spines have specialized shapes + vast numbers of proteins

b.   F-actin allows dendritic spines to change shape

Any info received at 1 dendritic spine, may have to reach a certain strength before being passed to dendrite to sent down cell body

-> initial step of computation that needs to occur when a cell receives info

e.g. to control synaptic vesicles, adhesion molecules that stick the pre + post synaptic side together, proteins found across whole synapse

Physiological role of dendritic spines (hint: 2 roles)

1.    Synapse formation

a.    Dendrite/ a protrusion can reach out

b.   Search surrounding neurons for appropriate pre-synaptic + partner + try to make connection

2.    Structural encoding of information

a.    Synaptic activity at axon

b.   Formation of a dendritic spine

c.    Information sent from pre to post

d.   Post synaptic side gets bigger -> connection strengthened -> LTP -> info is encoded, relevant for learning + memory

e.    Dendritic spine + post side formed under a different physiological stimulus

f.     -> e.g. neuromodulatory signal that could drive the formation of new dendritic spine

Does spinogenesis = synaptogenesis (imaging on graph → locations of both)

Dark area on imaging = post-synaptic density/PSD, juxtaposed to almost electron rich region on pre-synaptic side

Dendritic spines seen on border of dark area, can get dendritic spines without a presynaptic partner -> not making a synaptic connection -> distinguishes synaptogenesis from spinogenesis

Where are synaptic vesicles found?

Synaptic vesicles found at borders of pre + posy synaptic joint, PSD core found at postsynaptic border + PSD pallium below that

Synaptic vesicles location + what they allow

è Juxtaposed to electron rich side on pre synaptic

è Allows for synaptic vesicles to bind to active core + release content into synaptic cleft

è Majority of NT Rs found clustering around PSD (positioned in most effective way)

Key steps

Key steps in synaptogenesis (synapse formation) (4)

1.    Neurite Outgrowth & Guidance: Axons and dendrites extend toward each other, guided by chemical signals to find their target partners

a.    Post-synaptic side tries to find pre-synaptic partner, start to get target assembly

b.   long, thin protrusions of dendritic spines make connection

2.    Initial Contact/Adhesion: Specialized cell adhesion molecules (CAMs) bring pre- and postsynaptic membranes together

a.    when connection made, filipodia starts to change shape

3.    Synaptic Differentiation & Recruitment: Presynaptic components (vesicles) and postsynaptic components (receptors/scaffold proteins) are recruited to the contact site

a.    key proteins need to be first to come into a dendritic spine for it to become functional

4.    Refinement & Maturation: Activity-dependent mechanisms strengthen beneficial synapses and eliminate extraneous or weak ones

Key molecular mechanisms - Cell Adhesion Molecules (CAMs)

• Cell Adhesion Molecules (CAMs): Proteins like neurexins (presynaptic) and neuroligins (postsynaptic) ensure the connection is stable.

Key molecular mechanisms - Scaffold proteins

• Scaffold Proteins: Act as a "platform" to stabilize receptors and signaling machinery.

Key molecular mechanisms - Activity-dependent refinement

• Activity-Dependent Refinement: Ca2+ influx, often through NMDA receptors, strengthens functional connections, similar to Hebbian mechanisms of plasticity.

Key molecular mechanisms - Glia contribution

• Glia Contribution: Astrocytes and microglia play a key role in supporting and pruning synapses during this process

What are filopodia?

Filipodia – very dynamic + long dendritic protrusion -> different from dendritic spines (doesn’t contain many of the synaptic proteins you would expect to find in a fully functional dendritic spine)

How do dendritic spines work in surrounding neurons? (+ function of scaffold proteins here)

Dendritic spines are dynamic + protrusions will come out + search surrounding neurons for appropriate presynaptic partner -> if partner not found, will retract back into the dendrite/cell -> ready to go again + cell not losing anything

NMDAR, PSD95 (scaffold protein) + neurologind proteins are surfing around dendrite until they get a signal to move into dendritic spine -> where PSD should be -> start to make a connection

Synapse assembly + NMDAR function in it

Adhesion molecules, like neuroligands are important because they allow for actual physical connection (from pre + post synaptic side)

NMDAR = R that needs to be there to capture glutamate releases -> tells cell this should be afunctional dendritic spine

Synapse stabilisation (+ how adhesion mols help here)

è Involves recruitment of even more proteins to post-synaptic dendritic spine

è Includes an increase in the number of different types of adhesion proteins

Adhesion molecule on side of pre-synaptic: already in + out + about in presynaptic terminal, do not know if actually in active zone (adhesion protein shuttle up/down axon + hang around near presynaptic terminals + can be recruited also)

Testing the role of adhesion proteins: models to study syanpses

1.   In vivo – animal models -> knockout mice used to look at neural circuits +behavior

2.   In vitro – primary neuronal cultures, neurons taken e.g. from brain of newly born mouse/ embryonic rat, dissociated + plated -> cells grown in a dish to look at:

a.    Cellular effects

b.   Molecular/ biochemical effects

c.    Cortical pyramidal neuron (from rat embryonic brain)

 Experimental setup to study neurons in vitro (to study morphology of dendritic spines) - 4 steps

1.    DNA transfection to identify cell morphology, DNA plasmid (e.g. GFP/ green fluorescent protein), cortical neuron grown on glass coverslip

2.    Pharmacological treatment, fixing to stop dead at one point and look at morphology at that point in time

3.    Immunostaining with AB mixture, can use ABs raised against specific proteins to look at: distribution, abundance + localization of specific proteins

4.    Imaging using a microscope or live cell imaging

è Also can instead perfuse different types of chemicals + reagents, stimuli the cells however you want (on heated chamber with perfusion in + out)

è Add pharmacological treatment + fluorescent technique

Imaging of fixed cells – looking at morphology + localization of proteins

Analysis of dendritic spine morphology + synaptic protein localization/abundance:

-       Treatment/ genetic manipulation -> look for spine shrinkage, retraction, enlargement + formation

-       Can see movement of dendritic spines + postsynaptic protein at the same time

What are nascent synapses?

(Nascent synapses = developing, immature neural connections that bridge the gap between initial neuronal contact and fully mature functional synapses)

Role of Neuroligin 1 in recruiting PSD-95 to nascent synapses:

-       Hippocampal neurons, overexpressed green, fluorescent GFP tagged neuroligin 1

-       Analysis of synaptic protein localization/ abundance

o   +GFP-neuroligin1 (GFP-Nig1) -> treatment – clustering peptide + AB complex (using ICC-PSD95) -> analysis of protein abundance + spine morphology

-       Clustering: adhesion molecule joins clustering peptide + AB complex

N-cadherin ‘stabilises’ synapses

-       N-cadherin acts as a critical trans-synaptic, homophilic adhesion molecule that stabilizes synapse structure and regulates synaptic function

-       it is present at both pre- and postsynaptic sites

-       acting as a "zipper" that holds synaptic membranes together + acts as a scaffold for the underlying actin cytoskeleton

excitatory + inhibitory synaptic contact in different spine shapes

large dendritic spine -   excitatory synaptic contact

long, thin dendritic spine -  inhibitory synaptic contact

shapes of synapses in developing brain → mature brain phases (5)

Developing brain -> mature brain phases

-       filopodia

-       thin

-       stubby

-       mushroom

-       multi-headed

what components of a dendritic spine can provide information on its function? (2 + how)

the shape + size

→ spine size directly reflects synaptic strength

→ larger spines have more SA + scaffolding proteins to hold more AMPARs, making the synapse stronger

mushroom spines properties

• Mushroom (Large): Stable "memory" spines.

thin spines properties

• Thin (Small): Flexible "learning" spines. Low AMPA R levels, but primed to expand during LTP.

thin neck function

• Neck Function: The thin neck acts as a barrier, trapping AMPA receptors in the head to maintain signal intensity.

How LTP strengthens synapses by linking physical growth to more receptors (3 steps) - hint: how a small learning spine grows into large, AMPA-rich memory spine

1.    Calcium Influx: Ca2+ enters via NMDA receptors, activating the enzyme CaMKII.

2.    Actin Growth: CaMKII triggers rapid actin polymerization, physically pushing the spine membrane out to expand the head.

3.    AMPA Insertion: New AMPA receptors are shuttled to the surface and "locked" into the newly expanded scaffold (PSD-95).

Result: A small "learning" spine grows into a large, AMPA-rich "memory" spine

Organotypic slice cultures + glutamate uncaging (4 steps and process)

1.   Preparation

a.    Culture: Mature slices (7–14 DIV) on membrane inserts

b.   Transfection: Introduce fluorescent markers (e.g., GFP) via biolistics or AAV

c.    Perfusion: Move to chamber with oxygenated aCSF + caged glutamate (e.g., MNI-glutamate)

2.   Target Selection

a.    Visualization: Identify a clear dendritic branch using 2-photon imaging (920–930 nm)

b.   Focus: Zoom in on a single, mushroom-shaped spine

3.    Uncaging (The Pulse)

a.    Action: Trigger a second laser (720 nm) directed ~0.5 µm from the spine head

b.   Protocol:

                                              i.     Mapping: Single 1ms pulse

                                            ii.     Plasticity (sLTP): 30–60 pulses @ 0.5–2 Hz

4.   Measurement

a.    Morphology: Take Z-stacks every 1–5 mins to track volume growth

b.   Function: Use concurrent patch-clamp to record excitatory currents (uEPSCs)

Studying dendritic spines using glutamate uncaging

Studying dendritic spines using glutamate uncaging -   Glutamate uncaging induces spine formation (mechanism, de novo growth, input specificity + functionality)

a.    Mechanism: Repetitive uncaging of glutamate, often in low Mg2+ conditions or paired with depolarization, triggers the rapid (within minutes) enlargement of existing small dendritic spines.

b.   De Novo Growth: Glutamate uncaging can trigger the growth of brand-new, functional spines from the dendritic shaft, a process requiring NMDAR activation and PKA signaling, but not CAMKII or TrkB

c.    Input Specificity: These structural changes are strictly limited to the specifically stimulated spine, not neighbouring spines

d.   Functionality: Newly formed spines are functional almost immediately, expressing glutamate receptors and forming new, active synapses

Studying dendritic spines using glutamate uncaging -  High frequency stimulation (HFS) induces spine formation (structural LTP, size dependence + molecular requirements)

a.    Structural LTP: High-frequency electrical stimulation (e.g., 100 Hz) of excitatory inputs (Schaffer collaterals) mimics the structural changes induced by glutamate uncaging, resulting in lasting spine enlargement

b.   Size Dependence: HFS-induced growth preferentially affects smaller, more plastic spines rather than large, mature spines

c.    Molecular Requirements: Both HFS and glutamate-induced expansion depend on NMDA receptor activation and Ca2+ influx, which activate signalling cascades (like CaMKII) leading to actin polymerization

Linking dendritic spine structure with synaptic function

-       Dendritic spines act as specialized, individualized compartments for excitatory glutamatergic synapses, and their morphology is tightly coupled with synaptic function

-       A robust, positive correlation exists between dendritic spine head volume and the functional AMPA receptor (AMPAR) content at the postsynaptic density (PSD

Structural-Functional Correlation (in large spines/mushroom, small spines/thin/filopodia + stability/turnover)

a.    Large Spines (Mushroom): These contain larger PSDs, which have a higher capacity to hold AMPA receptors. Consequently, larger spines harbor more AMPARs, leading to stronger synaptic connections (higher sensitivity to glutamate)

b.   Small Spines (Thin/Filopodia): These contain fewer AMPARs and smaller PSDs, correlating to weaker synaptic strength

c.    Stability and Turnover: Larger, mature spines tend to be more structurally stable over months, while smaller spines are more plastic and have higher turnover rates

Linking Plasticity (LTP/LTD) (LTP, LTD + molecular link/GluR1)

a.    Long-Term Potentiation (LTP): Strong synaptic stimulation causes an influx of Ca2+ through NMDA receptors, promoting the insertion of new AMPARs into the synapse and inducing actin polymerization, which increases the spine head volume

b.   Long-Term Depression (LTD): Weak stimulation leads to AMPAR endocytosis (removal) and subsequent shrinkage of the spine head

c.    Molecular Link (GluR1): The AMPAR subunit GluR1 acts as a crucial link, where its insertion into the synaptic membrane not only strengthens transmission but its C-terminal tail helps stabilize the increased spine size

Functional Consequences (compartmentalisation + silent synapses)

a.    Compartmentalization: The narrow spine neck acts as a diffusion barrier, isolating the biochemical signalling (e.g. Ca2+ transients) within the spine head, ensuring that the structural changes are synapse-specific rather than influencing the entire neuron

b.   Silent Synapses: Small, thin spines may lack functional AMPARs, acting as "silent synapses" that can be matured into functional ones through the trafficking of receptors during potentiation

Structural plasticity of dendritic spines + relation to functional

Dendritic spines can change in shape in response to different stimuli:

1.    LTP – increase in size

2.    LTD – decrease in size

Synaptic activity (LTP/cLTP) -> synapse strengthening, more F-actin + increased synapse number but synaptic activity (LTD/cLTD has opposite effect)

Coordinating structural + functional plasticity study

-       Found that induction of chemical LTP (cLTP) caused spines to increase in size + amount of GluA1 also increased)

-       As spine size increased, amount of AMPA Rs also increased

-       Shows us that structural + functional plasticity are linked

Imaging dendritic spines in vivo (to image dendritic spines in thy1-YFP mice):

1.   Setup

a.    Preparation: Use a cranial window (permanent access) or thinned skull (minimally invasive)

b.   Fixation: Secure a metal head-plate to the skull to prevent motion blur

2.   Imaging

a.    Objective: High NA water immersion

b.   Depth: Focus on apical tufts in Layer 1 + capture Z-stacks

Structural plasticity – correlated with learning + memory?: Learning-Induced Spine Formation (hint: rapid remodelling, task-specific changes + correlation with performance)

a.    Rapid Remodeling: Training mice on new motor tasks (e.g., rotarod running) induces the formation of new dendritic spines on apical tuft dendrites of Layer 5 (L5) pyramidal neurons in the motor cortex

b.   Task-Specific Changes: The new spine formation is specific to the task learned, with different motor tasks resulting in structural changes on different dendritic branches

c.    Correlation with Performance: The extent of spine remodeling directly correlates with behavioral improvements, suggesting that structural plasticity is a direct measure of learning

Structural plasticity – correlated with learning + memory?:   Stabilization and Long-Term Memory (preference for stability, persistent memories + motor task specificity

a.    Preference for Stability: While many new spines are initially formed, a subset of them are preferentially stabilized after training, providing a structural basis for long-term memory

b.   Persistent Memories: These stabilized spines can persist for months, suggesting that learning leaves permanent structural marks on cortical connections

c.    Motor Task Specificity: New spines formed during learning are preferentially active when the mice perform the specific learned task, but not when they learn a new task, demonstrating their role in memory storage

Structural plasticity – correlated with learning + memory?: Age-Related Decline (reduced plasticity in older mice + accelerated elimination)

a.    Reduced Plasticity in Older Mice: Studies comparing young and old mice found that motor learning-dependent spine formation is reduced in the motor cortex of older mice, correlating with diminished learning ability

b.   Accelerated Elimination: Older mice show higher spine elimination rates during learning tasks compared to younger mice

Does dendritic spine structure link with synaptic function in vivo?

-       dendritic spine structure is linked with synaptic function in vivo

-       with sensory stimulation inducing NMDA receptor-dependent increases in surface GluA1 (sGluA1) that correlate with spine size

-       these findings indicate that in vivo structural remodelling of spines serves as a direct manifestation of experience-dependent synaptic strengthening

Spine shape (mushroom, thin + stubby) relation to when seen, used for what

è Mushroom Spines: Large heads, stable, and represent "memory" (stronger synapses)

è  Thin Spines: Smaller, highly motile, and represent "learning" (plastic synapses that can easily be strengthened or pruned)

è Stubby Spines: Often seen during early development, thought to be precursors or transitioning states

Whisker Stimulation Study (what it found, NMDAR dependence + activity-dependent trafficking)

-       Whisker Stimulation Study : In the somatosensory barrel cortex, acute sensory stimulation causes an increase in spine size and a concomitant increase in spine-surface GluA1-containing AMPA receptors (sGluA1)

-       NMDA Receptor Dependence: The increase in spine surface GluA1 evoked by stimulation is NMDA receptor-dependent, representing a form of in vivo LTP

-       Activity-Dependent Trafficking: The study demonstrated that the trafficking of GluA1 to the synapse is not static but changes in real-time in response to sensory experience

Coordinating structural + functional plasticity (hint: NDMAR/AMPAR and LTP/cLTP

NMDAR AMPAR -> synaptic activity (LTP/cLTP) -> enhanced AMPAR transmission, synapse strengthening + increased synapse number

-       NMDAR (The Signal): Detects activity and allows Ca2+entry

-       This calcium activates CaMKII, the master regulator for both processes

-       AMPAR (The Strength): Functional plasticity occurs when AMPARs are added to (LTP) or removed from (LTD) the membrane

-       The Link: Structural plasticity (spine growth) uses the actin cytoskeleton to expand the "docking space" for these AMPARs

-       Scaffolding: Proteins like PSD-95 act as physical slots, anchoring more AMPARs as the spine head grows

Mechanisms controlling structural and functional plasticity

-       primarily controlled by a network of actin-binding proteins (ABPs) and signaling molecules that regulate the polymerization, depolymerization, cross-linking, and stabilization of actin filaments (F-actin)

-       these mechanisms allow for rapid remodeling of actin networks in response to cellular signals, particularly in dendritic spines, where they mediate structural changes that align with functional synaptic changes like LTP + LTD

Kalirin-7 is required for Rac activity after NMDA-R activation:    Spine enlargement following cLTP (hint: essential role, NDMAR link + knockdown effects)

a.    Essential Role: Activated Kalirin-7 is necessary for NMDAR-dependent Rac1 activation, which drives actin polymerization and spine remodeling

b.   NMDAR Link: Kal7 interacts directly with the GluN2B subunit of NMDARs (via its PH domain) and acts as a GEF for Rac1

c.    Knockdown Effects: Inhibition or knockdown of Kalirin-7 blocks the activation of Rac1 and prevents subsequent spine enlargement following NMDAR stimulation

Kalirin-7 is required for Rac activity after NMDA-R activation: 1.    Kalirin-7 is phosphorylated following cLTP (hint: CaMKII activity + key residues)

a.    CaMKII Activity: Following cLTP, activation of NMDA receptors leads to the activation of CaMKII, which directly interacts with and phosphorylates Kalirin-7 in spines

b.   Key Residues: Phosphorylation of Kal7 by kinases such as CaMKII and Cdk5 at residues like T95 is a critical mechanism mediating its GEF activity and regulating spine size

Kalirin-7 is required for Rac activity after NMDA-R activation:    cLTP increase Rac activity via kalirin-7 (hint: mechanism, spine enlargement + essential for growth)

a.     Mechanism: cLTP induction triggers a signaling cascade: NMDAR -> CaMKII -> Kalirin-}7 (phosphorylation) ->  Rac1 (activation)

b.   Spine Enlargement: This pathway drives rapid spine enlargement and the insertion of GluR1-containing AMPA receptors into the synapse

c.    Essential for Growth: Without Kalirin-7 (e.g., in knockout models), the activity-dependent increase in spine volume and synaptic AMPA receptor recruitment is diminished or abolished

Kalirin-7 is required for Rac activity after NMDA-R activation:   kalirin-7 is found at synapses (hint: PSD localisation, scaffolding interaction + density regulation)

a.    PSD Localization: Kalirin-7 is the predominant adult isoform of the Kalrn gene and is highly enriched in the Postsynaptic Density (PSD) of dendritic spines in pyramidal neurons

b.   Scaffolding Interaction: It is anchored at the PSD through interactions with scaffolding proteins such as PSD-95, SAP-102, and SAP97

c.    Density Regulation: Its localization at the PSD is essential for its ability to regulate actin cytoskeletal dynamics and, consequently, synaptic function

What is Karlin-7 + what is its role in structional + functional plasticity at excitatory synapses?

-       Kalirin-7 (Kal7) = brain-specific neuronal guanine-nucleotide exchange factor (GEF)

-       plays a critical role in mediating the coordination of structural and functional plasticity at excitatory synapses, largely by regulating the actin cytoskeleton and receptor trafficking

How is Karlin-7 required for coordination of structural + functional plasticity

-       RNAi-mediated knockdown (KD) of Kalirin-7 in neurons leads to a reduction in dendritic spine density and impaired activity-dependent spine remodelling, highlighting its necessity for maintaining synapse structure

Role of Kal7 in structural + functional coordination

Kal7 connects NMDA receptor (NMDAR) activation to actin cytoskeletal rearrangements, enabling rapid enlargement of spines and strengthening of synaptic transmission

Kal7 mechanism of action

As a GEF, Kal7 activates the small GTPase Rac1. It is enriched in the postsynaptic density (PSD) where it interacts with PSD-95 and scaffolding proteins, enabling it to act as a signal integrator.

RNAi-mediated effects in Kal7

Studies using RNAi knockdown of Kal7 have demonstrated significant decreases in excitatory synapse density and spine loss

How does NMDAR control structural + functional plasticity

-       NMDA (The Gatekeeper): Acts as a coincidence detector. It stays blocked by magnesium until the neuron is sufficiently excited. Once open, it allows Ca2+ to enter

How does AMPAR control structural + functional plasticity

-       AMPA (The Strength): Responsible for the actual signal power. Their number on the surface determines how "strong" the synapse is

functional plasticity: LTP

LTP (Strengthen): High calcium influx -> Insertion of more AMPA receptors

functional plasticity: LTD

LTD (Weaken): Low calcium influx -> Removal of AMPA receptors

structural plasticity: growth

Growth: NMDA signals trigger the actin skeleton to expand the dendritic spine (larger "docking station")

structural plasticity: shrinkage

Shrinkage: Lack of activity leads to spine contraction or pruning

How are two ways a physiological stimulus can change? (From structural plasticity to refinement of neural circuitry) - and what this results in

o   the number of dendritic spines

o   the size of dendritic spines

-       This results in a change in the amount of AMPA receptors within the spines, resulting in a change of synaptic strength

-       Structural and functional plasticity are connected and can be changed, resulting in refined neural circuitry