# **Presynaptic Release Model: Sequence of Events & Multi-Timescale Modulations**

## **CORE RELEASE SEQUENCE (Milliseconds)**

### **Event 1: Action Potential Arrival**

```
Time: t = 0 ms
Trigger: Depolarization from axon hillock
Mechanism: Na⁺/K⁺ voltage-gated channel cascade
State: Terminal depolarizes from -70 mV to +30 mV
Duration: ~1 ms
```

### **Event 2: VGCC Opening**

```
Time: t = 0.2-0.5 ms after AP arrival
Trigger: Membrane depolarization > -40 mV
Types: CaV2.1 (P/Q-type), CaV2.2 (N-type) at active zone
Mechanism: Voltage sensor movement → pore opening
Open probability: ~0.3-0.8 during AP peak
Open duration: ~0.5-2 ms
```

### **Event 3: Ca²⁺ Influx & Microdomain Formation**

```
Time: t = 0.3-1 ms after AP arrival
Ca²⁺ source: Extracellular (1.2 mM) → intracellular (50 nM baseline)
Influx rate: ~3000-10000 Ca²⁺ ions per VGCC per ms
Microdomain formation:
  - Within 20 nm of VGCC: 10-100 µM
  - At vesicle release site: 10-25 µM threshold for release
  - Rise time: <100 µs
  - Diffusion-limited spread: ~100-200 nm radius
```

### **Event 4: Vesicle Release Decision**

```
Time: t = 0.4-1.5 ms after AP arrival
Decision conditions (AND logic):
  1. [Ca²⁺]microdomain > 10-25 µM
  2. Vesicle in RRP (docked & primed)
  3. SNARE complex in primed state
  4. Synaptotagmin Ca²⁺ binding sites occupied
Mechanism:
  - Ca²⁺ binds synaptotagmin (4+ cooperative sites)
  - Synaptotagmin displaces complexin clamp
  - SNARE complex completes zippering
  - Vesicle membrane fuses with presynaptic membrane
Release latency: 0.1-1.0 ms after Ca²⁺ threshold reached
Release synchrony: Multiple vesicles can release simultaneously
```

### **Event 5: Ca²⁺ Clearance**

```
Time: t = 1-50 ms after influx
Clearance mechanisms (in order of speed):
  1. Fast buffers (calbindin, parvalbumin): <1 ms
  2. Plasma membrane Ca²⁺ ATPase (PMCA): 10-100 ms
  3. Na⁺/Ca²⁺ exchanger (NCX): 10-100 ms
  4. Mitochondrial uptake: 10-1000 ms
  5. Endoplasmic reticulum uptake: 100-1000 ms
Residual Ca²⁺: 0.1-0.5 µM persists for 10-1000 ms
```

### **Event 6: Vesicle Recycling**

```
Time: t = 10 ms - 10 s (depending on activity)
Sequential steps:
  1. Endocytosis (clathrin-mediated, kiss-and-run, bulk)
  2. Vesicle re-acidification (v-ATPase)
  3. Neurotransmitter reloading (vesicular transporters)
  4. Priming (SNARE assembly, docking)
  5. Return to RRP
Recycling rate: Limited by ATP availability
```

## **MODULATION MATRIX ACROSS TIMESCALES**

### **MODULATION LEVEL 1: MILLISECONDS (Immediate Signal Processing)**

| Event            | Modulator                         | Effect                                 | Mechanism                                   | Timescale           |
|------------------|-----------------------------------|----------------------------------------|---------------------------------------------|---------------------|
| **VGCC Opening**     | Voltage waveform                  | AP width/duration affects open time    | K⁺ channel activation shapes repolarization | During AP (1 ms)    |
| **VGCC Opening**     | Ca²⁺-dependent inactivation (CDI) | Auto-inhibition                        | Ca²⁺ binding to calmodulin on VGCC          | 5-50 ms             |
| **Ca²⁺ Microdomain** | Endogenous buffers                | Shapes [Ca²⁺] spatial/temporal profile | Calbindin, parvalbumin binding              | <1 ms               |
| **Release Decision** | Synaptotagmin isoforms            | Different Ca²⁺ sensitivity             | Cooperative binding sites vary              | During Ca²⁺ binding |
| **Release Decision** | SNARE complex state               | Priming level determines speed         | Munc13 activity, NSF disassembly            | <1 ms               |
| **Ca²⁺ Clearance**   | Fast buffers                      | Immediate Ca²⁺ binding                 | High-affinity Ca²⁺ binding proteins         | <1 ms               |

### **MODULATION LEVEL 2: TENS-HUNDREDS OF MILLISECONDS (Short-term Plasticity)**

| Event               | Modulator            | Effect                        | Mechanism                                           | Timescale     |
|---------------------|----------------------|-------------------------------|-----------------------------------------------------|---------------|
| **VGCC Opening**        | Residual Ca²⁺        | Ca²⁺-dependent facilitation   | Ca²⁺/CaM binding to VGCC β-subunit                  | 10-1000 ms    |
| **VGCC Opening**        | eCB (CB1 activation) | Direct inhibition             | Gβγ subunit blocks pore                             | 100-2000 ms   |
| **Ca²⁺ Microdomain**    | Mitochondrial uptake | Buffer capacity changes       | Uniporter activity with Ca²⁺ buildup                | 10-1000 ms    |
| **RRP Replenishment**   | Residual Ca²⁺        | Enhanced mobilization         | Calcineurin activation → synapsin dephosphorylation | 10-1000 ms    |
| **RRP Replenishment**   | ATP availability     | Limits priming rate           | NSF, v-ATPase, priming ATPases                      | Continuous    |
| **Release Probability** | mGluR autoreceptors  | Activity-dependent inhibition | Gi/o → ↓cAMP → ↓PKA → ↓VGCC                         | 50-500 ms     |
| **Ca²⁺ Clearance**      | PMCA/NCX pumps       | Activity-dependent saturation | Limited pump capacity during high frequency         | 10-1000 ms    |
| **Vesicle Recycling**   | Activity level       | Different endocytosis modes   | High activity → bulk endocytosis                    | 100 ms - 10 s |

### **MODULATION LEVEL 3: SECONDS-MINUTES (Metabolic & Signaling)**

| Event             | Modulator                   | Effect                     | Mechanism                               | Timescale    |
|-------------------|-----------------------------|----------------------------|-----------------------------------------|--------------|
| **VGCC Density**      | Surface expression          | More/fewer channels        | Phosphorylation-dependent trafficking   | 30 s - 5 min |
| **VGCC Function**     | Kinase phosphorylation      | Changed open probability   | PKA, PKC, CaMKII phosphorylation        | 1-10 min     |
| **Ca²⁺ Dynamics**     | NO (cGMP pathway)           | Altered pump activity      | PKG phosphorylation of PMCA             | 1-10 min     |
| **Vesicle Pools**     | Mobilization rate           | RP→RRP speed changes       | Synapsin phosphorylation state          | 1-10 min     |
| **ATP Production**    | Lactate shuttle             | Enhanced ATP supply        | Astrocyte-neuron metabolic coupling     | 1-10 min     |
| **Release Machinery** | BDNF (acute)                | Enhanced priming & docking | TrkB → PLCγ → PKC activation            | 1-10 min     |
| **Autoreceptor**      | mGluR desensitization       | Reduced autoinhibition     | β-arrestin recruitment, internalization | 1-10 min     |
| **CB1 Receptor**      | eCB-induced desensitization | Reduced inhibition         | GRK phosphorylation, internalization    | 1-10 min     |
| **Ca²⁺ Clearance**    | Mitochondrial adaptation    | Increased capacity         | Matrix Ca²⁺ activates dehydrogenases    | 1-10 min     |

### **MODULATION LEVEL 4: MINUTES-HOURS (Structural & Transcriptional)**

| Event                 | Modulator          | Effect                        | Mechanism                                    | Timescale     |
|-----------------------|--------------------|-------------------------------|----------------------------------------------|---------------|
| **VGCC Expression**       | BDNF (chronic)     | Increased channel synthesis   | TrkB → MAPK → CREB → gene expression         | 30 min - 6 hr |
| **VGCC Expression**       | Activity-dependent | Homeostatic scaling           | Ca²⁺ → NFAT/CREB → VGCC genes                | 1-24 hr       |
| **Active Zone Size**      | BDNF, activity     | More release sites            | Structural proteins (RIM, Bassoon) synthesis | 1-12 hr       |
| **Vesicle Pool Size**     | BDNF, activity     | Larger RP/RRP                 | Synaptic vesicle protein synthesis           | 1-12 hr       |
| **SNARE Machinery**       | Local translation  | More fusion complexes         | mTOR-dependent protein synthesis             | 30 min - 3 hr |
| **Mitochondrial Density** | BDNF, activity     | Increased ATP capacity        | PGC-1α activation → biogenesis               | 3-24 hr       |
| **Metabolic Pathways**    | Activity patterns  | Enhanced glycolysis/oxidation | HIF-1α, AMPK pathway activation              | 1-6 hr        |
| **Receptor Expression**   | Chronic modulation | mGluR, CB1 level changes      | Transcriptional regulation                   | 3-12 hr       |
| **Buffer Expression**     | Ca²⁺ history       | Calbindin/parvalbumin levels  | Ca²⁺-dependent gene regulation               | 3-12 hr       |

### **MODULATION LEVEL 5: HOURS-DAYS (Metaplasticity & Structural Remodeling)**

| Event                     | Modulator             | Effect                              | Mechanism                                  | Timescale |
|---------------------------|-----------------------|-------------------------------------|--------------------------------------------|-----------|
| **Synapse Size**              | Chronic activity      | Growth/shrinkage                    | Cytoskeletal reorganization                | 6-48 hr   |
| **VGCC Distribution**         | Activity patterns     | Clustering changes                  | Scaffold protein (Homer, Shank) expression | 12-72 hr  |
| **Release Site Organization** | BDNF, activity        | Active zone architecture            | Piccolo, Bassoon, RIM expression           | 12-72 hr  |
| **Vesicle Recycling**         | Chronic patterns      | Efficiency changes                  | Endocytosis machinery expression           | 12-48 hr  |
| **Metaplasticity Rules**      | Activity history      | Altered LTP/LTD thresholds          | Kinase/phosphatase expression changes      | 24-72 hr  |
| **Energy Allocation**         | Chronic demand        | Mitochondrial positioning           | Motor protein (kinesin, dynein) adaptation | 12-48 hr  |
| **Network Integration**       | System-level activity | Presynaptic inhibition/facilitation | Interneuron connectivity changes           | 24-96 hr  |

## **MODULATION CASCADES (How Signals Propagate Through Events)**

### **Example 1: High-Frequency Activity Cascade**

```
Event Timeline:
t = 0 ms: First AP → Release (high Pr)
t = 10 ms: Second AP → Enhanced release (residual Ca²⁺ facilitation)
t = 100 ms: Tenth AP → Depressed release (RRP depletion, eCB inhibition)

Modulation Cascade:
Milliseconds: CDI develops, buffers saturate
Seconds: mGluR autoinhibition builds, ATP depletes
Minutes: BDNF synthesis begins, pumps adapt
Hours: VGCC expression increases, active zone grows
Days: Synapse strengthens, metaplasticity changes
```

### **Example 2: BDNF-Mediated Potentiation Cascade**

```
Event Timeline:
t = 0-5 min: BDNF binds TrkB receptors
t = 5-30 min: Kinase cascades activate
t = 30-120 min: Local protein synthesis
t = 3-24 hr: Structural changes

Modulation Cascade:
Milliseconds: Immediate VGCC phosphorylation (↑ open probability)
Seconds: Enhanced vesicle priming (↑ RRP replenishment)
Minutes: More SNARE complexes synthesized (↑ release sites)
Hours: Additional VGCCs inserted (↑ Ca²⁺ influx capacity)
Days: Active zone expansion (↑ number of release sites)
```

### **Example 3: eCB-Mediated Depression Cascade**

```
Event Timeline:
t = 100 ms: eCB produced postsynaptically
t = 200 ms: CB1 activation inhibits VGCCs
t = 1-10 min: β-arrestin recruitment, CB1 internalization
t = 1-24 hr: CB1 downregulation, structural changes

Modulation Cascade:
Milliseconds: Direct VGCC block (↓ Ca²⁺ influx)
Seconds: Reduced cAMP → ↓ PKA → ↓ vesicle priming
Minutes: CB1 internalization → reduced inhibition
Hours: CB1 downregulation → reduced modulatory capacity
Days: Synaptic downscaling → smaller active zone
```

## **CROSS-EVENT MODULATION PATTERNS**

### **Feedforward Loops:**

```
Positive:
AP → Ca²⁺ influx → CaMKII activation → ↑ VGCC phosphorylation → ↑ Ca²⁺ influx

Negative:
AP → Glutamate release → mGluR activation → ↓ VGCC opening → ↓ Ca²⁺ influx
```

### **Feedback Loops:**

```
Homeostatic:
High activity → Ca²⁺ buildup → gene expression → more buffers/pumps → ↓ Ca²⁺ buildup

Trophic:
Activity → BDNF release → structural growth → more release sites → more activity
```

### **Energy Management Loops:**

```
Energy Conservation:
ATP depletion → AMPK activation → ↓ protein synthesis → ↓ energy consumption

Energy Investment:
BDNF + adequate ATP → mTOR activation → protein synthesis → structural growth
```

## **INTEGRATED TIMELINE OF A RELEASE EVENT WITH MODULATIONS**

### **t = 0-1 ms (AP to Release)**

```
Primary events: AP → VGCC open → Ca²⁺ influx → Release
Modulations active:
  - AP shape from previous activity (K⁺ channel adaptation)
  - VGCDI from recent Ca²⁺ history
  - SNARE priming from recent ATP availability
  - Buffer capacity from expression levels
```

### **t = 1-100 ms (Immediate Aftermath)**

```
Primary events: Ca²⁺ clearance, vesicle recycling initiation
Modulations active:
  - Residual Ca²⁺ facilitating next release
  - mGluR autoinhibition building
  - eCB production if postsynaptic Ca²⁺ high
  - ATP consumption for pumps and recycling
```

### **t = 100 ms - 1 min (Short-term Adaptation)**

```
Primary events: RRP replenishment, metabolic adjustments
Modulations active:
  - Kinase/phosphatase balance shifting
  - NO production if sustained activity
  - BDNF release if activity pattern significant
  - Mitochondrial Ca²⁺ uptake affecting ATP
```

### **t = 1 min - 1 hr (Medium-term Plasticity)**

```
Primary events: Protein synthesis, trafficking changes
Modulations active:
  - Local translation of VGCCs, SNAREs
  - Receptor trafficking to/from surface
  - Mitochondrial positioning changes
  - Metabolic pathway adjustments
```

### **t = 1 hr - days (Long-term Restructuring)**

```
Primary events: Structural changes, gene expression
Modulations active:
  - Nuclear signaling for gene expression
  - Cytoskeletal reorganization
  - Synapse growth/shrinkage
  - Metaplasticity rule changes
```

## **MODELING PRINCIPLES**

1. **Hierarchical Modulation**: Faster events constrain slower adaptations
2. **Bidirectional Coupling**: Every modulation affects and is affected by release events
3. **Energy Constraints**: ATP availability gates all processes
4. **History Dependence**: Past activity patterns determine current modulation states
5. **Context Specificity**: Same modulator can have opposite effects depending on timing/context
6. **Redundancy**: Multiple pathways can achieve similar outcomes
7. **Trade-offs**: Every enhancement has costs (energy, stability, flexibility)

This model shows how a simple millisecond release event sits at the center of a rich, multi-timescale regulatory network where each release both responds to and influences modulations across seconds to days, creating a dynamic, self-tuning system for synaptic transmission.