I am currently working with paired patch clamp recordings between CB1+ basket cells and pyramidal cells in CA1. I use coronal slices from young adult mice (28-42 days old), cut in sucrose-based aCSF, 300 um, and then incubated in normal aCSF for 1 hour before recordings.
One problem I am experiencing is the huge variability in tonic endocannabinoid signalling at these synapses, as measured by the success rate of IPSCs following presynaptic stimulation. I use trains of 50 APs at 10 Hz as presynaptic stimulation, once per minute. Only very few pairs show the expected 70% success rate (as previously seen by Soltesz lab), while most show barely any failure. I am sure of the interneurons identity as I do all the anatomical characterizations later on.
I was wondering if anyone has experienced this before, and if there are any tips and tricks I should be aware of?
Thanks in advance!
What is the temperature and external Calcium you use? Endocannabinoid release is dependent on these factors. I would use low EGTA in the postsynaptic cell. We saw quite a bit of variability in the calyx of Held synapse. Only half of our cells responded to DHPG (a mGluR1/5 agonist that elicited Endocannabinoid release in our postsynaptic cells and subsequent CB1-medited presynaptic Ca current inhibition; See Kushmerick et al., JNeurosci. 2004). i would try to follow the exact recipes of other groups like Soltesz lab...
Thanks for your reply. I'm using the exact recipes from the Soltesz lab, both for intra and extracellular solutions. Extracellulal calcium is 2.5 mM and temperature is 33-34 °C.
Hi Marco,
I spent a long time looking for CB1+Interneuron->Pyr connected pairs in both hippocampus and cortex. For these experiments I was using the GAD65 GFP mice from Gabor Szabo where you can find some CB1+ IN, among many others (I guess you are patching blind). CB1+ IN where in most of the cases regular spiking neurons with basket morphology. I studied these connections a bit more in detail in L2/3 of the cortex and basically what you describe in your post: high variability (from 100% to 25% of failures). In any case, regardless the initial failure rate, Am251 application consistently increased the efficiency of transmission. The "awakening" of this synapses with presynaptic AP trains was successful only in connections with low initial probability of release and required long time (>3 minutes) to fully recover. Bottom line, I also observed a high heterogeneity in CB1+ connections that I would attribute to both pre and postsynaptic elements.
Hope this helps.
Thanks Pablo,
good to know that you were also experiencing similar results. I guess I'll just keep going then!
Regards,
Marco
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