# Presentation: 435901

## Presynaptic actions of endocannabindoids

**Presynaptic actions of endocannabindoids**

**Bradley E. Alger**

## Acknowledgements

**Darrin Brager**
**Greg Carlson**
**Jimok Kim**
**Sergei Kirov**
**Fiona LeBeau**
**Rob Lenz**
**Masako Isokawa **
**Wade Morishita**
**Tom Pitler**
**Chris Reich**
**Namita Varma**
**John Wagner**

**Catherine Ledent**
**Zoya Katerova**
**Frank Margolis**

**Paul Luther**
**Jon Lederer**
**Scott Thompson**

## Recording ConditionsBrain slice preparation

**Extracellular**
**Solution (mM****)**
**NaCl****	****120**
**KCl ****	****    3 **
**MgSO****4**** ****	****    2 **
**NaH****2****PO****4****	****      ****1**
**Glucose****	****  10**
**CaCl****2****	**** 2.5**** **
**APV****	****0.05**
**NBQX****	****0.01**

**Recording Pipette**
**Solution (mM)**
**CsCH****3****SO****3              ****100**
**CsCl                     50**
**Hepes                  10**
**CaCl****2****	****   0.2/0.1/0**
**BAPTA****	****2.0/1.0/0.2**
**Mg****2****ATP****	****              2**
**KCl                        1**
**MgCl****2****                    1**
**QX314                   2**
**TEACl****	****             10**

**Temp = 28 – 30****o**** C **
**Osm = 290-310 mOsm**
**pH = 7.20 - 7.25**

**GABA**

** ****PYR**

**Notes:**
-to isolate inhibitory post-synaptic currents, or IPSCs, I include APV to block NMDA receptors and NBQX to block AMPA and kainate receptors
-this knocks out ionotropic excitatory glutamatergic tranmission, thus leaving me with a monosynaptic IPSC when I stimulate in s. r. near the Inns
-I record from the postsynaptic CA1 pyramidal cell in whole-cell voltage clamp at resting potential (-70)
-voltage clamp again is a technique to control the voltage from the cell I am recording from, the pyr cell, while simultaneously measuring currents in that cell
-then I stimulate the presynaptic cell, which releases GABA into the cleft, which in turn binds to GABAA receptors to elicit an IPSC

-in the recording pipette, I include high Cl to increase the driving force for the IPSCs; in other words, when GABA binds to its receptors and the pore opens, Cl goes out of the cell whic h is seen as a downward deflection or an inward current in voltage clamp
-cesium and TEA which non-selectively block voltage-dependent and leak K channels, and QX-314 to block Na channels and inwardly rectifying  K channels 
-so blocking Na channels functionally removes the postsynaptic axons; leaving only events that occur in the soma or dendritic region of the cell
-the resulting response then is  a monosynaptic evoked GABAA-mediated IPSC
-and when we give a depolarizing step to this CA1 pyr cell, a retro messenger is released which acts to decrease the activity of this IN cell-this decrease in activity is termed DSI...

## Depolarization-induced Suppression of Inhibition (DSI) 

**20 s**

**Current**
**   ****(pA)**

**Time (ms)**

**0**

**-1000**

**-2000**

**0**

**100**

**200**

**D****epolarization-induced ****S****uppression of ****I****nhibition (****DSI****)**

**Notes:**
-DSI affects both spontaneous and evoked IPSCs, but I will mainly focus on the evoked IPSCs
-here is a representative example of evoked DSI
-first is  a plot of current vs. time
-I am stimulating every 3 seconds, again, downward deflections are monosynaptic evoked IPSCs, giving a DSI step every 90 seconds from -70 mV to 0 mV, for a duration of 2 seconds
-you can see a transient  reproducible reduction in the eIPSC amplitude that lasts roughly 30 s to 1 minute, depending on the trial
-below you can see the same downward deflections on a faster timescale
-the first trace is an average of 8 IPSCs immed preceding the step
-the second trace is an average of 5 IPSCs immed following the step
-as you can see, this is signif smaller after the step

-so, again, this is a representative example, showing a transient reduction in postsynaptic IPSCs after a depol step
-we have adopted a quantitative method for assessing DSI...

## Endocannabinoids mediate DSI/DSE

**CB1 receptors on certain presynaptic neurons.**

**Ca****2****+**

**CB1**

**2-AG (?) **

**Endocannabinoids mediate DSI/DSE**

## Slide 6

## Slide 7

## Activation of CB1Rs inhibits exocytosis

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**Time (s)**

**Activation of CB1Rs inhibits exocytosis**

**2 Hz**

## The Model

**The Model**

## X

**IN**

**4**

**2**

**3**

**1**

**X**

**Presynaptic actions of endocannabinoids**

**Post**

## Endocannabinoids inhibit   presynaptic Ca2+ influx

**Endocannabinoids inhibit   presynaptic Ca****2+ ****influx**

## Endocannabinoids inhibit quantal release

**Endocannabinoids inhibit quantal release**

**Diana and Marty, J. Neuroscience, July, 2003**

## Blocking certain K+ channels blocks DSI

**20 s**

**200 pA**

**50 s**

**200 pA**

**Control**

**4-AP**

**D**

**D**

**sIPSCs**

**Blocking certain K****+ ****channels blocks DSI**

**Varma, et al., Neuropharm, Sept., 2002**

**D**

**D**

**D**

**D**

## Endocannabinoids inhibit neuronal firing

**Endocannabinoids inhibit neuronal firing**

**Kreitzer, Carter and Regehr, Neuron, May 2002**

## Short-range and long-range 

**actions of endocannabinoids**

**IN**

**Endocannabinoid**

**From Kreitzer and Regehr, Neuron, May, 2002**

## X

**IN**

**4**

**2**

**3**

**1**

**X**

**Presynaptic actions of endocannabinoids in DSI/DSE**

**Post**

## Activation of CB1 can cause long-term depression (LTD)

**Activation of CB1 can cause long-term depression (LTD)**

**CB1****+/+**

**CB1****-/-**

## Endocannabinoids induce LTD after 	prolonged CB1R activation

**Endocannabinoids induce LTD after ****	****prolonged CB1R activation**

**From Chevaleyre and Castillo, Neuron, May, 2003**

**mGluR stimulation**

**AM-251**

## Prolonged CB1R activation alone is not sufficient to cause LTD

**Prolonged CB1R activation alone is not sufficient to cause LTD**

**Kim and Alger, Soc Neurosci Abstr, Nov 2003**

## Summary

**DSI and DSE are produced by short-term retrograde action of endocannabinoids on presynaptic cells.**
**Endocannabinoids inhibit Ca****2+ ****influx, increase K****+**** efflux and interfere with the quantal release process.**
**Prolonged activation of CB1Rs can cause long-term depression of glutamate or GABA release.  (Other factor?)**

## The Model

**The Model**

## Endocannabinoids and CB1R

- O
- ||

**Ca****2+**

- 2-AG

- NH

- anandamide

- membrane lipid bilayer

- postsynapticcell interior

- Synaptic cleft

- lipases and diesterases

**CB1R**

- presynapticcell interior

- O
- ||

- O

- HO

**1**

**2**

**3**

**4**

**5**

**degradation**

**reuptake**

## C

**C**

- Time (s)

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**Normalized intensity**

**0.0**

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**1.0**

**1.2**

- 5 Hz

**B**

**Time (s)**

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**Intensity (a.u.)**

**0**

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- Baseline

- Steady-state

- 5 Hz

- Fitting

## Slide 24

## Endocannabinoids inhibit Ca2+ influx

**Diana, Levenes, Mackie, and Marty, J Neurosci, Jan, 2002 **

**Endocannabinoids inhibit Ca****2+**** influx**

## A

** ****CTX**

**500 nM**

- n = 546

- Amplitude (pA)

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- Total # of events

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- n = 314

- Amplitude (pA)

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- n = 546

- Amplitude (pA)

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- Total # of events

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- n = 1625

- 0

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- # of events

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- n = 1050

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- n = 1719

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**Baseline**

**DSI**

**Recovery**