Posts

Showing posts from August, 2014

Nomeclature regarding AAV constructs

Q:  "On UNC vector core website (and DREADD wiki pages), I found two types, AAV2/5/8-CAMIIKa-HA-HM3Dq-IRES-mCitrine or AAV8-hsyn-HM3Dq-mCherry.

My question is, which one is better? and can I know the difference between them, in the aspect of expression level or anatomical specificity?

A:  The nomneclature gives the answer:

AAV2/5/8-CAMIIka-HA-hM3Dq-IRES-mCitrene=

This is available in three serotypes (AAV2, AAV5 and AAV8)=we recommend starting with AAV8 serotypes 


CAMKIIka=the promotor. In this case the promotor is selective for neurons which express CAMIIKa (e.g. glutamatergic neurons in general)

HA=Hemagluttinin tag

hM3Dq=Gq DREADD

IRES=internal ribosome entry site

mCitrene=GFP variant which is quite bright and more resistant to photobleaching than GFP


hSyn=human synapsin promotor which in this case specifies neuronal expression

M3Dq-mCherry=Gq DREADD as mCherry fusion protein

Thus, both will express Gq DREADD in neurons but one will specify excitatory neurons.



BRAIN Initiative Video on-line

A new video has been posted today on the BRAIN initiative which is quite informative regarding notions about where we are, where we need to go and so on.

DREADD technology is highlighted at the 3hr 20 min (or so) time point--particularly as it relates to translating DREADD technology to non-human primates and on-going work optimizing novel DREADDs and non-CNO-based ligands.

Question re: retrograde transport of AAV serotypes

A user asks:

" Publications indicate that AAV5 has a preferential retrograde transport among AAV types. Have you had this same experience?"

A:  My lab has no experience.

There are, though, several recent papers which have looked into this in mouse brain where AAV5 was retrogradely transported, in rat cortical neurons in culture,  in non-human primate where AAV6 is reported to be retrogradely transported, as well as AAV6 inrats appears to be retrogradely transported.

Others with experience are welcome to add comments and/or advice.

Bryan

DREADDs in non-human primates

There is a series of three abstracts now on-line for the SFN2014 meeting relating what appears to be a very comprehensive analysis of DREADDs as tools to interrogate circuits, behavior and perception in non-human primates (NHP).

The first of the abstracts apparently uses 11C-CNO as a PET ligand to validate hM4Di expression in vivo which is quite nifty.

The abstracts are listed below and may be accessed via thison-line tool.

 Program#/Poster#:462.22/TT58Presentation Title:In vivo PET imaging of the behaviorally active designer receptor in macaque monkeys
Program#/Poster#:462.24/TT60Presentation Title:Reversible DREADD inactivation of less than 10% of Orbitofrontal cortex neurons in interconnection with rhinal cortex is sufficient to disrupt cue discrimination in monkeysProgram#/Poster#:462.23/TT59Presentation Title:Reversible DREADD inactivation of orbitofrontal cortex neurons in rhesus monkeys with contralateral rhinal cortex removal disrupts cued reward discrimination. I. Behavioral a…

Deconstructing memory with DREADDs

There are now quite a few papers using DREADDs in an inducible fashion to deconstruct the memory trace, block memoriesand augmentmemory.

I note that there are some intriguing differences with certain experiments and parallel studies done with optogenetics.  Among the various possibilities that have been suggested (see this review), it is important to remember that DREADD-based approaches utilize conserved and canonical modes of signaling to augment or diminish neuronal firing in an asynchronous fashion while optogenetics approaches essentially induce an artificial, synchronized firing pattern.

Should I make up CNO fresh or use frozen stocks?

Question:  I have a quick question about preparation of CNO. CNO will be prepared as 0.1 mg/ml in 0.25%DMSO/0.9% saline, and I am wondering whether this solution can be stored in 4C for a long term or should be stored in -20C (or should be prepared each day before using).

Answer: We recommend making CNO fresh before each use.