Optogenetics, a neuromodulation method that is employed to control and monitor the activities of individual neurons in living tissue and was first developed by researchers Edward Boyden and Karl Deisseroth in 20051, is now considered as being one of the main pillars of neuroscience research.
In 2010, the journal Nature Methods choose optogenetics as the ‘Method of the Year’2 across all fields of science and engineering. In the same year the academic research journal Science highlighted it in the article ‘Breakthroughs of the Decade’3. The basis of this technique is to genetically modify neurons to express light-sensitive ion channels such as excitatory channelrhodopsin or inhibitory halorhodospin and then use light to control the on/off status of neuronal excitation. Great progress has been made with this technology since its invention and today even conscious free-moving animal models can be manipulated and observed in real-time.
Gene Targeting Optogenetics In Rats Is Now A Reality
Rats have long been used as models for neuroscience research due to their higher intelligence. Yet a prerequisite of the application of optogenetics technology in any animal models is the ability to target the expression of opsin genes in desired populations of neurons. This is conventionally achieved by employing the Cre-LoxP system4, which depends on two lines of genetically modified animals: a conditional opsin expression line in which the opsin expression cassette is under the control of a universal promoter but preceded by a floxed stop cassette; and a Cre line that expresses the phage recombinase Cre from a promoter that provides spatial or temporal control over where and when the floxed stop cassette could be excised, which leads to the expression of the opsin gene.
Fortunately, with the advent of programmable nuclease technologies such as zinc finger nuclease and CRIPSR/Cas 9 system, the technical hurdle to introduce the Cre-LoxP system to the rat has been overcome5 , which may usher in the return of the once popular rat models in the neuroscience field.
A Targeted Optogenetic Approach
Utilizing zinc finger nuclease or CRISPR/Cas9 technology, a suite of knock-in rat models6 for the neuroscience research community, consisting of various neuron-specific Cre drivers, Cre activity-dependent fluorescent reporter and opsin-expressing rat lines has been Created and is now available commercially. A prominent feature of all Cre lines in this toolbox is that the Cre gene is inserted immediately downstream of the endogenous locus of targeted genes. This is in great contrast to most available rat Cre lines established so far, which are produced via random transgenes and bear the risk of possible positional effects as well as incomplete inclusion of regulatory elements of a promoter, leading to undesired expression patterns. The targeted integration of the Cre coding sequence into an endogenous locus should lead to the expression of Cre from the genomic context of the target gene, potentially with the same pattern.
Indeed, preliminary analysis of these commercially available knock-in Cre rat models demonstrate the faithful expression of the Cre recombinase. For example, the Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and will have the overarching ability to control neuronal activity using optogenetics especially in the study of Parkinson’s disease.
Similarly, the conditional opsin expressing lines, in which the conditional expression cassette of opsin is targeted to the Rosa26 locus, show expected opsin-fluorescent fusion protein expression upon being mated with various Cre lines.
Future Applications For Optogenetics
Where can optogenetics take us? So far, the majority of research utilizing optogenetics technology is carried out with genetically modified mouse models, thanks to the availability of hundreds of tissue-specific Cre lines. Whereas such studies have contributed enormous knowledge to our understanding on how the brain works and how neurological diseases occur, further studies with better models are necessary or even indispensable. For example, rats have been found to be adept at episodic memory7, which is among the first to be lost in patients with Alzheimer’s disease. Considering the high failure rate of drugs for Alzheimer’s disease tested in mice, rat models may out-perform mouse ones. Now with the Creation of the optogenetic toolbox in the rats, such studies could possibly be shifted to rats, whose brain may better mimic the human brain.
It is also worth to note that while this toolbox offers the option to conditionally express either excitatory (ChR2H134R) or inhibitory (eNpHR3.0) opsin in various neuron subtypes (~10) by simply mating a Cre line and an opsin expression line, its application could be greatly expanded by combining with either Cre expressing virus to drive opsin expression in different subtypes of neurons or Cre-dependent opsin expressing virus to achieve the expression of opsin with different channel properties8. In addition, the application of the toolbox may not be limited to the central nervous system. After all, the toolbox is not the limit. Our imagination is.
Gene targeting strategy
Knock-in rats produced
|Cre||CamK2a-Cre||LEH||Calcium/Calmodulin-dependent protein kinase II alpha positive neurons|
|Cre||DAT-Cre||SD&LEH||Dopamine Active Transporter positive (dopaminergic) neurons (Liu et al, 2016)|
|Cre||HTR3A-cre||LEH||5'-hydroxytryptamine receptor 3A positive serotonergic neurons|
|Cre||Pvalb-Cre||LEH||Paralbumin positive GABAergic interneurons|
|Cre||Slc32A1(VGAT)-Cre||LEH||Slc32A1 positive GABAergic interneurons|
|Cre||Sst-Cre*||LEH||Somatostatin positive neurons|
|Cre||Th-Cre**||SD||Tyrosine Hydroxylase positive (dopaminergic) neurons (Liu et al, 2016)|
|Cre||Tph2-Cre||LEH||Trypotophan Hydroxlyase 2 positive serotonergic neurons|
|Cre||VIP-Cre||LEH||Vasoactive Intestinal Polypeptide positive GABAergic interneurons|
|Cre||NPY-Cre*||LEH||Neuropeptide Y positive neurons|
|Reporter||Rosa Tom||LEH||Cre activity-dependent Tdtomato reporter|
|Optogenetics||Rosa ChR2H134R-EYFP* Channelrhodopsin*||LEH||Cre activity-dependent Tdtomato reporter|
|Optogenetics||Rosa NpHR3.0-Tom Halorhodopsin||LEH||Cre activity-dependent Tdtomato NpHR3.0-Tdtomato fusion protein expression|
*Will be commercially available soon
**Female Th-Cre rats could be used as a germ line deleter to derive whole body knock-out rats from conditional knockout rats.
- Millisecond-timescale, genetically targeted optical control of neural activity
- Method of the Year 2010.
- Stepping Away From the Trees For a Look at the Forest
- Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons. Liu Z, et al. PLOS One 2016
- Whole-rat conditional gene knockout via genome editing
- Introduction to a genetic toolbox for neuroscience: neuron-specific Cre, fluorescent Cre activity reporter and conditional optogenetics knock-ion rats. Z. Liu and G. Zhao (Horizon Discovery). Poster presented at SFN17.
- Rats that reminisce may lead to better tests for Alzheimer’s drugs
- The form and function of channelrhodospin