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Showing posts from 2014

DREADDs in newborn neurons

I occasionally receive queries regarding the use of retrovirally delivered DREADDs to newly born neurons in dentate gyrus.  Here is a nice use of this technology along with optogenetics in hippocampus in Neuron.

Using intersectional genetics and DREADDs to deconstruct breathing

Very nice paper out in Cell Reports on this topic.

A note about authorship, MTAs, IP and other matters related to DREADDs

IP:

As should be evident to all (though I still get queries about this) DREADD technology is open source with no associated intellectual property (IP)-encumbrances. There is no need for an MTA to use/obtain the technology*.

The technology is not patented and is given freely to the scientific community without restrictions as to use.  Future enhancements also will never be patented by me.

This means that if you make a 'non-obvious enhancement' you conceivably could patent it.

What this means is that you can obtain the *cDNAs from ADDGENE, *mice from JAX and viruses from UNC and use them for whatever purposes you desire without permission from my lab.  This includes pharma/biotech.

Publications:

As a general rule not include me or any member of my lab as authors on your papers using DREADD technology simply because we shared a reagent with you or offered you helpful advice (e.g. how to dissolve CNO).  If you are unsure, please discuss with us before you submit the paper (or send …

DREADDs, fast-spiking interneurons and animal models of schizophrenia

I get periodic queries as to whether/if hM4D can inhibit the firing of fast-spiking interneurons.  The answer is, apparently, yes.


The authors go on to make connections with circuitry dysregulation posited to occur in schizophrenia...

Hypothalamic regulation of energy balance deconstructed using DREADDs

Interesting paper in this vein published in Nature Neurosciences today.  Useful information related to the time-course of CNO's effects which might be  useful to others which are transient.

Lentiviral Vectors

We occasionally receive requests for the lentiviral vectors in Alexander et al, Neuron 2009.  We will soon make them available via ADDGENE but for now simply contact Hu ( from the Rothlab

Happy Halloween from the Roth lab

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Manipulating a cocaine 'engram' chemogenetically

Pretty amazing paper now on-line from the Josselyn/Frankland labs showing that

"...Ablating or silencing neurons overexpressing CREB (due to cocaine) before testing disrupted the expression of a previously acquired cocaine memory, suggesting ... a critical hub in what is likely a larger cocaine memory engram."


DREADD review now on-line

A comprehensive review with many useful tables of DREADD applications is now on-line.

Floxed DREADD mice

I get many requests for these.

The Rothlab may have such beasts in hand and will make them available via JAX as soon as they are validated. Rather than sending emails to that lab directly, as soon as the mice are deposited in JAX it will be announced here.

DREADDs in rats redux

An elegant paper in Cell today demonstrating (again) that local infusions of CNO can modulate terminal release in rats.  Again, TH-Cre rats were used and both excitatory and inhibitory DREADDs were used to modulate norepinephrine release and thereby modify choice decisions.

Where can I obtain CAV-Cre for 'retro-DREADD'-esque studies

I see that this facility in France may provide CAV-Cre

http://www.biocampus.cnrs.fr/index.php/en/plateformes?id=78


How much virus should I inject?

Q: "I have some Gi-DREADDs that I got from UNC and am preparing to inject an aliquot of them into the cortex.

Can you tell me if the sample I got from UNC should be diluted or should it be used as is?  If diluted, what do you recommend and how much?"

A1: "We look at the titer and generally inject 10^9/uL--so it may need to be diluted to 10^9/ul"
Explanation: we try to inject virus with a titer generally ~ 10^9/ul
A2:  "It depends on what kind of experiments you are going to do. Some experiments you need to dilute the virus just like Bryan said. Some are not.
You can aliquot the virus into 5ul/tube after thawing the virus on ice, and store it in -80. Try your injection with different dilutions of virus. Check the expression and then decide what dilution is good for you. "

DREADDs using cell-type specific promotors

Q: "Doyou know of anyone who has put DREADD into locus coeruleus?"

A: yes a very nice paper recently using PRSx8 and lentivirus.  Per the authors:

 "PRSx8 is based on an upstream regulatory site in the human DBH promoter and drives high levels of expression in adrenergic neurons"


Use of AAV for therapy of human CNS diseases: relevance for use of DREADDs in humans

The recent posts on the use of DREADDs in primates and novel lentiviral approaches in primate brainhas raised the issue of the suitability of virally-mediated transduction of human neurons for delivery of various cargos (including DREADDs).

Clinicaltrials.gov shows many ongoing/completed trials of AAV-based gene delivery for a variety of CNS diseases in humans indicating that this is certainly feasible.

Targeting primate neurons with lentiviral vectors

A very nice technical paper has appearedwhich describes many of the parameters which can be optimized to achieve cell-type specific expression of many cargos (including DREADDs) in primates using lentiviral vectors.

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.  

DREADDs for deconstructing signaling pathways in cellular processes

Two nice papers by the Gutkind lab (NIH) have used DREADDs in a synthetic biology context for deconstructing signaling pathways involved in processes in triple negative breast cancer (Science Signaling) and in mitogenic processes (Molecular Cell).

The paper in Molecular Cell is particularly interesting (IMHO) because it lays out a strategy whereby DREADDs as a synthetic biology platform are combined with genome-wide RNAi to identify down-stream signaling nodes and networks.

One can imagine such an approach being particularly useful in model organisms such as Drosophila and C. elegans as well as utilizing CRISPR-based strategies for genome-wide interrogation of functionalities.

Probably y'all can imagine other utilities...

DREADD-assisted metabolic mapping (DREAMM)

An interesting and potentially useful technology combining DREADDs and microPET imaging has been described recently.  Essentially it allows for identification of circuits involved in various behaviors in freely moving animals.

Genotyping protocols for DREADD-expressing mice--where to find them?

Protocols and other detailed items are generally found on the DREADD resource wiki page

If there is a protocol you would like (or like to see edited) please let us know.

DREADDs, feeding and a useful Cre-driver line

There are now many papers demonstrating bidirectional, cell-type-specific control of feeding behavior using DREADDs (see Sternson and Roth, in press and Wess et alfor reviews) so why highlight yet another one?

Here theauthorscreate a useful new Cre-driver line (orexin-Cre) which will may be useful.  Of note apparently only 30% of neurons are essential for driving behavior....

DREADD-mediated control of IPS-derived neurons in vivo

An interestingpaper out today that I've been hearing about regarding bidirectional control of transplanted DA neurons in a Parkinson's Disease model.

Video primer on DREADD technology

DREADDs in astrocytes

There are now papers appearing (and more in the near future most likely) where hM3Dq has been used to "activate" astrocyte Ca++ release.

In some cases the effects are quite striking andsuggest a pleiotropic role of astrocytes in regulating neuronal activity throughout the neuraxis.

There also is a nice paper suggesting a role of astrocytic Ca++ in ETOH self-administration.


How best to dissolve CNO ...it depends on what it is soluble in (and that depends on the particular polymorph)

I recently received the following question:

"My question is with regard to the use of CNO in drinking water (in mouse studies). Your recipe calls for the addition of a small amount of DMSO to dissolve CNO at a concentration of 5 mg in 200 mL. As we and others routinely dissolve CNO in 0.9% saline (no DMSO) at concentrations up to four times this amount, I am curious whether the addition of DMSO to CNO solutions for H2O drinking is critical, or whether the properties of CNO permit it's direct dissolution in H2O at concentrations from 5-20 mg per 200 mL. There appears to be no difficulty dissolving such concentrations in H2O in my hands, but I would prefer to hear your perspective. Is there a published reference for the solubility profile of CNO in H2O or 0.9% saline?"

It basically depends upon the source of the CNO.  CNO via the NIH RAID program (which is what we use) is not readily soluble in H20 while commercial CNO is.  Although we've verified that both sources are …

Alexander 'Sasha' Shulgin ==RIP

For those of you who are interested in psychopharmacology and it's history, I note the passing ofAlexander 'Sasha' Shulgin.  Although he was not active in science for many years in the 1960's he published several intriguing structure-activity papers in Nature and elsewhere ...

DREADD amelioration of seizures; synaptic silencing redux

A provocative new paper on-line inNature Communications shows hM4Di when activated by CNO suppresses a variety of experimental seizures.  Also further evidence for silencing of synapses with hM4Di in this paper....

DREADDs in rats

I occasionally get questions regarding approaches for using DREADDs in rats.  There are now a number of papers wherein cell-type specific expression of DREADDs in neurons was achieved using several platforms:

HSV-transduction using either Enkephalin or Prodynorphin promotors for cell-type specific expression in striatal neurons:Transient neuronal inhibition reveals opposing roles of indirect and direct pathways in sensitizationFerguson SM, Eskenazi D, Ishikawa M, Wanat MJ, Phillips PE, Dong Y,Roth BL,NeumaierJF.Nat Neurosci. 2011 Jan;14(1):22-4. doi: 10.1038/nn.2703. Epub 2010 Dec 5.AAV-DIO-DREADD using TH-Cre miceNorepinephrine-neuron-selective expression  using AAV-based delivery and the synthetic promoter PRSx8

DREADDs in Drosophila

I get occasional queries from folks in Pharma and Academia about model organisms and using DREADDs for various genetic/chemical genetic screening platforms.

A suite of DREADD-based flies are available fromCharles Nichols and published in Cell Reports.  I believe Chuck will make these available (or may already) at Flybase.

Of course they work great inyeast and probably as well in all other organisms which use GPCRs (fish, worms, llamas, humans).

Bryan

Engineering chemotaxis with DREADDs

A cool application of DREADD technology is found in a nice paper from the Wendall Lim lab at UCSF.

This elegantly demonstrates how synthetic biology tools may be used to modulate cell motility (in this case chemotaxis) in the periphery.  It is likely that this approach will prove useful for all types of motile cells.


DREADD-induced presynaptic silencing

This is something I've been hearing about for some time and is now on-line in Neuronfrom Stachniac, Ghosh and Sternson wherein they nicely demonstrate that hM4Di induces presynaptic silencing (via an unknown but likely interesting mechanism).

They also report an hM4D-neurexin variant which targets to axons.  We also have such a beast which may have improved surface expression and transport which may be similarly useful and is now available to all who wish to use it.  I see the Sternson variant is now available via ADDGENE.

This will be a highly useful reagent for spatio-temporal control of synaptic transmission. I note that Steve Mahler from Gary Aston-Jones' lab has also reported in Nature Neuroscience what appears to be a similar observation related to presynaptic dopamine release via local infusion of CNO.

A useful approach for activation of specific neuronal pathways using DREADDs and CAV-1 Cre

A nice proof-of-concept paper was just published in PLOS ONE

Combined Use of the Canine Adenovirus-2 and DREADD-Technology to Activate Specific Neural Pathways In Vivo

Here canine associated adenovirus (CAV) expressing Cre-recombinase was used to specifically express hM3Dq (AAV-DIO-hSyn-hM3Dq-mCherry) in VTA neurons projecting to the striatum.  This paper is nice as it describes the technology in some detail.

I note a similar use of this approach was previously described by Carter et al (Nature, 2013).

My sense is that this technology will be broadly useful...

New DREADD constructs and controls available from ADDGENE

A large batch of new plasmids (25) are now available viaADDGENE






CNO via minipump

I've received several queries regarding the delivery of CNO via Alzet (or similar) minipumps.

As far as I'm aware no data using this as a delivery platform for CNO has been published (nor has my lab attempted this as we typically simply put CNO in the drinking water (http://chemogenetic.blogspot.com/2014/03/cno-in-drinking-water.html)

Nonetheless--it should work.  Anyone with success/failure feel free to add comments.


CNO in drinking water

We frequently get requests regarding protocols for putting CNO in the drinking water for long-term studies.

Here is our protocol:

1. Dissolve the CNO in a small volume of DMSO
2. Diluted the dissolved CNO in drinking water (e.g., 5mg/200ml)
3. Give mice with CNO drinking water  and protect from light using foil-wrapped bottles
4. The mice will receive 5mg/kg/day CNO (assume that mice weight 30g and consume 6ml water per day)
5. Prepare fresh daily.
6. A small amount of saccharine in the drinking water will mask the slightly bitter taste of CNO

IRES constructs redux

I've received quite a few emails regarding my decision to no longer offer high titer viral stocks of the various IRES constructs via the UNC vector core.  And thought I'd clarify for those interested....

The constructs work beautifully in our hands (see for instance our recent paper where we used the CAMKII-hM4Di-IRES-mCitrene vector) and yield highly reproducible and high levels of transduction in mice.  

I have received a small number of emails from users who have not been able to reproducibly achieve high levels of transduction when the DIO-hSyn-DREADD-IRES-mCitrene constructs are used in combination with a Cre-driver line (even though they function well in our hands).

My sense is that there was some variability on the packaging on the part of the vector core and I directed them to take them off line.

The plasmids are/will be available from ADDGENE.

Bryan

Recommended strategy for anti-HA staining

We've characterized a number of antibodies and piloted many protocols for visualizing HA-tagged DREADDs.

Here's our recommended protocol (please note suppliers of antibodies for best results):

Immunofluorescence Staining Protocol (Anti-HA antibody)
1. Perfuse the mice with ice cold 4% paraformaldehyde (PFA; freshly prepared)

Cryosection procedure:
2. Fix the brain with 4%PFA @ 4°C overnight (O/N).
3. Transfer brain sample to 30% sucrose/1x phosphate buffered saline (PBS), allow sample sink @ 4°C for about 48hr.
4. Cryosection 40 uM

Staining procedure:
5. Permeabilize the tissue with 1xPBS/0.4% Triton x100 for 1hr at room temperature (RT).
6. Block the slide with 1% BSA 5% NGS (normal goat serum) in 1xPBS/0.4% Triton x100 at RT for 1hr.
7. Incubate the sections with primary antibody (1:500) in blocking buffer @ 4 O/N.
8. Wash the section with 1x PBS/0.4% Triton x100 three times at RT, 10min/each.
9. Incubate the sections with secondary antibody (1:250) in blocking buffer for 2hr a…

Recommended starting constructs for new DREADD users

For those of you using AAV-based delivery of DREADDs we recommend that you consider using the following constructs/viruses if you are using rodents.

Non-Cre-dependent

AAV8-hSyn-hM3Dq-mCherry
AAV8-hSyn-hM4Di-mCherry


Cre-dependent

AAV8-DIO-hSyn-hM3Dq-mCherry

AAV8-DIO-hSyn-hM4Di-mCherry

We've found these give excellent transduction in a variety of brain regions and high levels of expression in neurons.

Also, if you fix the tissue please consider using an anti-mCherry antibody rather than endogenous mCherry fluorescence to visualize the DREADD-mCherry fusion protein as considerable quenching of fluorescence occurs following fixation.

Bryan

IRES constructs

The Rothlab (http://pdsp.med.unc.edu/rothlab/) has received feedback from users regarding various DIO DREADD-based IRES constructs (e.g. DIO-hSyn-DREADD-IRES-YFP).  Our sense is that because of the large size of the insert, AAV-based packaging is suboptimal leading to low transduction efficiency.
Accordingly we are no longer offering AAV viral stocks of these vectors.  
If users wish to obtain the cDNAs they will soon be available via addgene:
https://www.addgene.org/Bryan_Roth/
Bryan
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This is a blog to provide feedback and comments from users regarding chemogenetic technologies.