Guan et al, Intl J Obesity, 2002.

Different types of neurons are interdigitated in the same brain areas:

How selectively stimulate/inhibit?

Davidson & Breakefield, Nat. Rev. Neurosci. 2003.

Gene transfer in brain in vivo

viral vectors

Calloway, E., Trends Neurosci. 2005.

Cell-type-specific gene transfer & modulation of

neuronal activity

Allostatin (AL) = invertebrate peptide/receptor system

Evolving the lock to fit the key: Designer

receptors exclusively activated by designer

drugs (DREADDS)

Conklin, B., PNAS (2007)

Armbruster et al., PNAS (2007)

directed evolution: muscarinic ACh receptor -> DREADD

CNO=clozapine n-oxide

Muscarinic Ach receptor

DEADD

Courtesy of Bryan Roth, U North Carolina

Specific neuronal inhibition with hM4D DREADD

Lentivirus to drive DREADD via orexin promoter: selective activation or inhibition of orexin

neurons in behaving animals

WPRE

ampR

HIV Sequence

HIV LTR'

hORX p

EGFP

LTRPsi

IRES

DREADD

SOWVG-D13880 bp

•Brain orexin/hypocxretin system

• Arousal, stress• Reward, addiction• Eating, obesity

Photosensitive ion channels for control of neuronal activity

Zhang et al., Nature Rev. Neurosci. (2007)

Channelrhodopsin Halorhodopsin

excitation inhibition

Photostimulation yields more specific effects than electrical stimulation

Alternative to Deep Brain Stimulation

Zhang et al., Nature Rev. Neurosci. (2007)

Cell-type specific expression of channelrhodopsin in brain norepinephrine neurons in vivo

PRSx8 promoter; mCherry tagMicroinjection in vivo in locus coeruleus

TH (NE neurons) mCherry (ChR2 neurons) Merge

David MoormanElena Vazey

Photostimulation of CNS neurons in vivo

record laser

Photostimulation of neuronal activity in vivo

Locus coeruleus neuronal recordings in anesthetized rat

David MoormanElena Vazey

Summary

• Novel proteins designed and expressed in neurons• Designer receptors• Photosensitive channels/ion pumps

• Neurons transduced in vivo to express novel proteins• Viral vectors (lenti virus, adeno-associated virus, etc)• Cell-type specific expression using promoters

• Many clinical applications• Designer receptors (DREADDS): More specific than conventional pharmacology• Photostimulation: More specific, less toxic than Deep Brain Stimulation (DBS)• Much better targeting ability than conventional approaches, e.g., DBS, lesion• Many fewer side-effects

Acknowledgements

Rachel SmithDavid MoormanAngie CasonPouya Tashili-FahadanKimberlei RichardsonSteve MahlerLana ZhangGreg SartorElena VazeyMike SmithBecky FallonGlenda Harris (U Penn) Matt Wimmer (U Penn)

MUSC Neuroscience of Addiction Research Center

National Institute on Drug AbuseLilly Pharmaceuticals

1414

Transfer Vector DesignTransfer Vector Design

PromotersPromoters– mPGK-1mPGK-1– hSyn-1: neuronshSyn-1: neurons– hGFAP: gliahGFAP: glia– CaM kinase IICaM kinase II– rMORrMOR– rPPErPPE

Internal ribosome entry sites (IRES)Internal ribosome entry sites (IRES)– Encephalomyocarditis virus (ECMV)Encephalomyocarditis virus (ECMV)– Synthetic (Gtx)Synthetic (Gtx)

Woodchuck hepatitis virus post-translational Woodchuck hepatitis virus post-translational regulatory element (WPRE)regulatory element (WPRE)

Promoter Transgene IRES EGFP WPRE