Cre-dependent DREADD (Designer Receptors Exclusively Activated by Designer Drugs) mice: Conditional DREADD Mice

DREADDs, designer receptors exclusively activated by designer drugs, are engineered G protein-coupled receptors (GPCR) which can precisely control GPCR signaling pathways (for example, Gq, Gs and Gi). This chemogenetic technology for control of GPCR signaling has been successfully applied in a variety of in vivo studies, including in mice, to remotely control GPCR signaling, for example, in neurons, glia cells, pancreatic beta-cells, or cancer cells. In order to fully explore the in vivo applications of the DREADD technology we generated hM3Dq and hM4Di strains of mice which allow for Cre recombinase-mediated restricted expression of these pathway-selective DREADDs. With the many Cre driver lines now available, these DREADD lines will be applicable to studying a wide array of research and preclinical questions

Non-invasive activation of optogenetic actuators

The manipulation of genetically targeted neurons with light (optogenetics) continues to provide unprecedented avenues into studying the function of the mammalian brain. However, potential translation into the clinical arena faces a number of significant hurdles, foremost among them the need for insertion of optical fibers into the brain to deliver light to opsins expressed on neuronal membranes. In order to overcome these hardware-related problems, we have developed an alternative strategy for delivering light to opsins which does not involve fiber implants. Rather, the light is produced by a protein, luciferase, which oxidizes intravenously applied substrate, thereby emitting bioluminescence. In proof-ofprinciple studies employing a fusion protein of a light-generating luciferase to a light-sensing opsin (luminopsin), we showed that light emitted by Gaussia luciferase is indeed able to activate channelrhodopsin, allowing modulation of neuronal activity when expressed in cultured neurons. Here we assessed applicability of the concept in vivo in mice expressing luminopsins from viral vectors and from genetically engineered transgenes. The experiments demonstrate that intravenously applied substrate reaches neurons in the brain, causing the luciferase to produce bioluminescence which can be imaged in vivo, and that activation of channelrhodopsin by bioluminescence is sufficient to affect behavior. Further developments of such technology based on combining optogenetics with bioluminescence - i.e. combining lightsensing molecules with biologically produced light through luciferases - should bring optogenetics closer to clinical applications.Published versio

Preserved functionality of individual moieties within luminopsin-1.

<p>(A) HEK cells were transfected with native, secreted Gaussia luciferase (GLuc), luminopsin-1 (LMO1; GLuc-ChR2 fusion gene), or with ChR2 alone and bioluminescence was determined by adding CTZ to the medium or to the cells. Only secreted GLuc produced signal in the medium, while bioluminescence generated by LMO1 was found only in cells. (B) Comparison of the bioluminescence signals obtained from cells (10⁴ cells per well, 4 wells per group) transfected with native, secreted GLuc (GLuc), luminopsin-1 (LMO1, GLuc-ChR2 fusion construct), and Renilla luciferase (Renilla) as well as untransfected cells (None). Bioluminescence was comparable between native GLuc and GLuc within LMO1. Luminescence from LMO1 and Renilla was significantly different (*p<0.05; one-way ANOVA followed by Tukey’s test). (C) Comparison of photocurrents induced by physical light in HEK cells transfected with ChR2 (upper panel) and LMO1 (GLuc-ChR2; lower panel). (D) Luminance-photocurrents curves for ChR2 and LMO1 (GLuc-ChR2). When normalized, they showed similar half-maximal luminances (1/2 max). n = 3. (E) No significant differences in maximum photocurrents between ChR2 and LMO1 (GLuc-ChR2) were observed (p>0.05; two-tailed Students’ T-test; n = 3 each). Error bars denote S.E.M in this and subsequent figures.</p

Modulation of neuronal activity by luminescence.

<p>Hippocampal neurons were transfected with LMO2 (GLuc-VChR1) and their intrinsic excitability was examined before and after CTZ application. (A) A square pulse (150 pA; top) was injected to a hippocampal neuron under current clamp, eliciting action potential firing (middle). After CTZ application, the neuron fired more action potentials (bottom). (B) Spike frequency with 150-pA current injection was significantly increased by CTZ (*p<0.05; two-tailed paired Students’ T-test; n = 3). (C) Prerecorded subthreshold spontaneous excitatory postsynaptic currents (sEPSCs; top) were injected into a neuron under current clamp, inducing single action potential (control; second from top). After CTZ application action potential firing was increased (CTZ 2 s; third from top) and returned to the baseline (CTZ 30 s; bottom). (D) On average, CTZ application transiently enhanced action potential firing up to 50%; n = 7.</p

Design of luminopsins, luciferase-channelrhodopsin fusion proteins.

<p>Gaussia luciferase (GLuc) is fused to the N-terminus of channelrhodopsin (ChR). Yellow fluorescent protein (YFP) is fused to the C-terminus of ChR. Application of the GLuc substrate coelenterazine (CTZ) leads to an enzymatic reaction resulting in the production of light (photons) and opening of the channel.</p

Luminescence-evoked responses in neurons.

<p>Hippocampal neurons were transfected with LMO2 (GLuc-VChR1) and patch-clamped (A). Expression of the fusion protein was visualized by YFP fluorescence (B). Upon CTZ application, GLuc generated bioluminescence throughout the processes (C). This bioluminescence evoked both inward current under voltage-clamp (D) and depolarization under current-clamp (E).</p

Altered Behavior in Mice Socially Isolated During Adolescence Corresponds With Immature Dendritic Spine Morphology and Impaired Plasticity in the Prefrontal Cortex

Mice socially isolated during adolescence exhibit behaviors of anxiety, depression and impaired social interaction. Although these behaviors are well documented, very little is known about the associated neurobiological changes that accompany these behaviors. It has been hypothesized that social isolation during adolescence alters the development of the prefrontal cortex, based on similar behavioral abnormalities observed in isolated mice and those with disruption of this structure. To establish relationships between behavior and underlying neurobiological changes in the prefrontal cortex, Thy-1-GFP mice were isolated from weaning until adulthood and compared to group-housed littermates regarding behavior, electrophysiological activity and dendritic morphology. Results indicate an immaturity of dendritic spines in single housed animals, with dendritic spines appearing smaller and thinner. Single housed mice additionally show impaired plasticity through measures of long-term potentiation. Together these findings suggest an altered development and impairment of the prefrontal cortex of these animals underlying their behavioral characteristics

Luminescence activated photocurrent.

<p>LMO1 (GLuc-ChR2)-expressing HEK cell (A) was identified by YFP fluorescence (B) and patch-clamped. Coelenterazine (CTZ) application near the cell elicited bioluminescence (C). (D) Luminescence (upper trace) and luminescence-induced photocurrent (lower trace) were recorded simultaneously from the same cell.</p