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Showing posts from 2015

DREADDs in 2015

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In 2015 there were 358 papers where DREADD technology played an important role.  I created a WORDLE using these and it looks like (no surprise) Neurons were used as frequent targets.

My top picks for 2015:

1.Chemogenetic disconnection of monkey orbitofrontal and rhinal cortex reversibly disrupts reward value.
Eldridge MA, Lerchner W, Saunders RC, Kaneko H, Krausz KW, Gonzalez FJ, Ji B, Higuchi M, Minamimoto T, Richmond BJ. Nat Neurosci. 2016 Jan;19(1):37-9. doi: 10.1038/nn.4192. Epub 2015 Dec 14.

2.  A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior.
Vardy E, Robinson JE, Li C, Olsen RH, DiBerto JF, Giguere PM, Sassano FM, Huang XP, Zhu H, Urban DJ, White KL, Rittiner JE, Crowley NA, Pleil KE, Mazzone CM, Mosier PD, Song J, Kash TL, Malanga CJ, Krashes MJ, Roth BL. Neuron. 2015 May 20;86(4):936-46. doi: 10.1016/j.neuron.2015.03.065. Epub 2015 Apr 30

3.  The first structure-activity relationship studies for designerreceptorsexclusively activated bydesigner drugs…

JCI: Chemogenetic (and other approaches) interrogate inflammation-associated aversion in DA neurons

Systemic inflammation causes malaise and general feelings of discomfort. This fundamental aspect of the sickness response reduces the quality of life for people suffering from chronic inflammatory diseases and is a nuisance during mild infections like common colds or the flu. To investigate how inflammation is perceived as unpleasant and causes negative affect, we used a behavioral test in which mice avoid an environment that they have learned to associate with inflammation-induced discomfort. Using a combination of cell-type–specific gene deletions, pharmacology, and chemogenetics, we found that systemic inflammation triggered aversion through MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Further, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor–expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic …

Nice review by Sternson et al on technologies for deconstructing appetite

Cell Metabolism

Advances in neuro-technology for mapping, manipulating, and monitoring molecularly defined cell types are rapidly advancing insight into neural circuits that regulate appetite. Here, we review these important tools and their applications in circuits that control food seeking and consumption. Technical capabilities provided by these tools establish a rigorous experimental framework for research into the neurobiology of hunger.

Nicely balanced view of all the current technological approaches.

Happy Birthday to our resident wizard

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Using chemogenetics to deconstruct G12/13 signaling in lymphoma

Another interesting paper from theGutkind lab

NATURE COMMUNICATIONS: Chemo- and optogenetic dissection of ventral hippocampus modulation of feeding

Previous research has focused on feeding circuits residing in the hindbrain and midbrain that govern homeostatic or hedonic control of food intake. However, the feeding circuits controlling emotional or cognitive aspects of food intake are largely unknown. Herewe use chemical genetics and optogenetic techniques to dissect appetite control circuits originating from ventral hippocampus (vHPC), a brain region implicated in emotion and cognition. We find that the vHPC projects functional glutamatergic synaptic inputs to the lateral septum (LS) and optogenetic activation of vHPC projections in LS reduces food intake. Consistently, food intake is suppressed by chemogenetic activation of glutamatergic neurons in the vHPC that project to the LS and inactivation of LS neurons blunts vHPC-induced suppression of feeding. Collectively, our results identify an anorexigenic neural circuit originating from vHPC to LS in the brain, revealing a potential therapeutic target for the treatment of anorex…

NATURE NEUROSCIENCE: DREADDs in primates

Here's the first of likely many of these papers --has some interesting proof-of-concept findings as well as caveats related to CNO in primates.  For those interested in DREADDs in primates you may find our new CNO analogues more useful.

MOLECULAR PSYCHIATRY: Opto- and chemogenetic interrogation of ketamine actions--

Very cool and comprehensive paper; again use chemogenetic not pharmacogenetic

A single sub-anesthetic dose of ketamine exerts rapid and sustained antidepressant effects. Here, we examined the role of the ventral hippocampus (vHipp)-medial prefrontal cortex (mPFC) pathway in ketamine’s antidepressant response. Inactivation of the vHipp with lidocaine prevented the sustained, but not acute, antidepressant-like effect of ketamine as measured by the forced swim test (FST). Moreover, optogenetic as well as pharmacogenetic specific activation of the vHipp–mPFC pathway using DREADDs (designer receptors exclusively activated by designer drugs) mimicked the antidepressant-like response to ketamine; importantly, this was pathway specific, in that activation of a vHipp to nucleus accumbens circuit did not do this. Furthermore, optogenetic inactivation of the vHipp/mPFC pathway at the time of FST completely reversed ketamine’s antidepressant response. In addition, we found that a transient increas…

NEURON: using DREADDs to elucidate cholinergic signaling vis a vis Parkinson's Disease

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Nice paper --with the key chemogenetic experiment shown below and nice use of ChAT Cre




THE SCIENTIST: review of chemogenetic technology

Remote Mind Control Using chemogenetic tools to spur the brain into action By Kelly Rae Chi|November 1, 2015

Here.

CELL REPORTS: Bidirectional modulation of Raphe neuronal activity by DREADDs

A paper which confirms and extends our recent findings using FLOXED-DREADD mice rather than AAV-DIO-DREADD approach.

I note the term pharmacogenetic rather than chemogenetic but otherwise interesting.

"Despite the well-established role of serotonin signaling in mood regulation, causal relationships between serotonergic neuronal activity and behavior remain poorly understood. Using a pharmacogenetic approach, we find that selectively increasing serotonergic neuronal activity in wild-type mice is anxiogenic and reduces floating in theforced-swim test, whereas inhibition has no effect on the same measures. In a developmental mouse model of altered emotional behavior, increased anxiety anddepression-like behaviors correlate with reduced dorsal raphé and increased median raphé serotonergic activity. These mice display blunted responses to serotonergic stimulation and behavioral rescues through serotonergic inhibition. Furthermore, we identify opposing consequences of dorsal versus medi…

CELL: DREADDS for deconstructing serotonergic components of innate and learned fear

Fear is induced by innate and learned mechanisms involving separate pathways. Here, we used an olfactory-mediated innate-fear versus learned-fear paradigm to investigate how these pathways are integrated. Notably, prior presentation of innate-fear stimuli inhibited learned-freezing response, but not vice versa. Whole-brain mapping and pharmacological screening indicated that serotonin-2A receptor (Htr2a)-expressing cells in the central amygdala (CeA) control both innate and learned freezing, but in opposing directions. In vivo fiber photometry analyses in freely moving mice indicated that innate but not learned-fear stimuli suppressed the activity of Htr2a-expressing CeA cells. Artificial inactivation of these cells upregulated innate-freezing response and downregulated learned-freezing response. Thus, Htr2a-expressing CeA cells serve as a hierarchy generator, prioritizing innate fear over learned fear.

SCIENCE: chemogenetics and neuronal ablation to deconstruct itch; plus a new hM4Di mouse

From the Goulding lab:

Mice in which dorsal NPY::Cre-derived neurons are selectively ablated or silenced develop mechanical itch without an increase in sensitivity to chemical itch or pain. This chronic itch state is histamine-independent and is transmitted independently of neurons that express the gastrin-releasing peptide receptor. Thus, our studies reveal a dedicated spinal cord inhibitory pathway that gates the transmission of mechanical itch.

NATURE: Combining the awesome power of yeast with computation to discover new ligands for GPCRs

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Not a chemogenetic paper but a chemical genetic paper which again proves that yeast are most awesome!

Thanks to Ethan Perlstein for the graphic

At least 120 non-olfactory G-protein-coupled receptors in the human genome are ‘orphans’ for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach le…

Chemo- and opto-induced remapping of place cells

The pharmacologically selective designer Gi-protein-coupled muscarinic receptor hM4D or the light-responsive microbial proton pump archaerhodopsin (ArchT) was expressed in MEC, and place cells were recorded after application of the inert ligand clozapine-N-oxide (CNO) or light at appropriate wavelengths. CNO or light caused partial inactivation of the MEC. The inactivation was followed by substantial remapping in the hippocampus, without disruption of the spatial firing properties of individual neurons. The results point to MEC input as an element of the mechanism for remapping in place cells.

Paper here

Chemogenetic Deconstruction of Basal forebrain control of wakefulness and cortical rhythms--Nature Communications

Wakefulness, along with fast cortical rhythms and associated cognition, depend on the basal forebrain (BF). BF cholinergic cell loss in dementia and the sedative effect of anti-cholinergic drugs have long implicated these neurons as important for cognition and wakefulness. The BF also contains intermingled inhibitory GABAergic and excitatory glutamatergic cell groups whose exact neurobiological roles are unclear. Here we show that genetically targeted chemogenetic activation of BF cholinergic or glutamatergic neurons in behaving mice produced significant effects on state consolidation and/or the electroencephalogram but had no effect on total wake. Similar activation of BF GABAergic neurons produced sustained wakefulness and high-frequency cortical rhythms, whereas chemogenetic inhibition increased sleep. Our findings reveal a major contribution of BF GABAergic neurons to wakefulness and the fast cortical rhythms associated with cognition. These findings may be clinically applicable t…

Useful resource for kinase experiments

Going forward it will be useful to identify the signaling nodes downstream of chemo- and optogenetic perturbations of neuronal circuits.  A nice paper has appeared which characterizes a comprehensive set of kinase inhibitors.

Amazing DREADD paper in Science today

A quite amazing paper has just appeared:

Cells of a common developmental origin regulate REM/non-REM sleep and wakefulness in mice
Mammalian sleep comprises rapid eye movement (REM) sleep and non-REM (NREM) sleep. To functionally isolate from the complex mixture of neurons populating the brainstem pons those involved in REM/NREM sleep switching, we pharmacogenetically manipulated neurons of a specific embryonic cell lineage in mice. We identified excitatory glutamatergic neurons that inhibit REM sleep and promote NREM sleep. These neurons shared a common developmental origin with neurons promoting wakefulness, both derived from a pool of proneural hindbrain cells expressingAtoh1 at embryonic day 10.5. We also identified inhibitory GABAergic neurons that act downstream to inhibit REM sleep. Artificial reduction or prolongation of REM sleep in turn affected slow wave activity (SWA) during subsequent NREM sleep, implicating REM sleep in the regulation of NREM sleep.

Just a note to folks go…

Chemogenetic manipulation of ventral pallidal neurons impairs acquisition of sign-tracking in rats

Interesting paper here.

DREADD -based deconstruction of brain response to 'simulated dawn'

Fascinating paper way outside my area of expertise using a variety of techiques to address question of "Twice a day, at dawn and dusk, we experience gradual but very high amplitude changes in background light intensity (irradiance)."

Book of DREADDs

A protocol book devoted to DREADD technology has now appeared and it looks useful

BNST controls alcohol seeking--modulation with DREADDS

DREADD-induced silencing disrupts the preference of female mice for opposite-sex chemosignals

Interesting paper from SFN's new on-line journal

DREADDs to deconstruct histamine action in the periphery

Interesting paper here.

New chemogenetics review

Here:

Neurologic disorders are frequently a result of inappropriate electrical and/or chemical signaling of neurons and glia. Ultimate remediation would necessitate reprogramming these signals. Historically, correcting neuronal and glial signaling is accomplished via drug therapy/administration, although they frequently fail to effectively and fully treat the underlying disorder. Developments in basic research have produced several new classes of potential therapeutics to directly and precisely control neuron activity at the single-cell level. We review one such technology, Designer Receptors Exclusively Activated by Designer Drugs, and suggest its potential as a powerful tool for augmenting neuronal and glial signaling and activity for basic and translational applications.


DREADDs to treat Parkinson's Disease

Interesting paper here.

Background Patients with advanced Parkinson's disease (PD) often present with axial symptoms, including postural- and gait difficulties that respond poorly to dopaminergic agents. Although deep brain stimulation (DBS) of a highly heterogeneous brain structure, the pedunculopontine nucleus (PPN), improves such symptoms, the underlying neuronal substrate responsible for the clinical benefits remains largely unknown, thus hampering optimization of DBS interventions. Choline acetyltransferase (ChAT)::Cre + transgenic rats were sham-lesioned or rendered parkinsonian through intranigral, unihemispheric stereotaxic administration of the ubiquitin-proteasomal system inhibitor, lactacystin, combined with designer receptors exclusively activated by designer drugs (DREADD), to activate the cholinergic neurons of the nucleus tegmenti pedunculopontine (PPTg), the rat equivalent of the human PPN. We have previously shown that the lactacystin rat model accurately reflect…

Elucidating actions of DRN 5HT neurons on behavior and circuits with DREADDS

Elucidating how the brain’s serotonergic network mediates diverse behavioral actions over both relatively short (minutes-hours) and long period of time (days-weeks) remains a major challenge for neuroscience. Our relative ignorance is largely due to the lack of technologies with robustness, reversibility and spatio-temporal control. Recently we have demonstrated that our chemogenetic approach (eg, Designer ReceptorsExclusively Activated by Designer Drugs, DREADDs) provides a reliable and robust tool for controlling genetically defined neural populations. Here we show how short- and long-term activation of dorsal raphe nucleus (DRN) serotonergic neurons induces robust behavioral responses. We found that both short- and long-term activation of DRN serotonergic neurons induce antidepressant-like behavioral responses. However, only short-term activation induces anxiogenic-like behaviors. In parallel, these behavioral phenotypes were associated with a metabolic map of whole brain network a…

GPCR-ome resource now available from ADDGENE

Our published resource now now available via ADDGENE which allows for interrogation of essentially all of the human druggable GPCR-ome.

DREADDs to deconstruct cocaine actions

Additionally, using designer receptors exclusively activated by designer drugs (DREADDs) technology, we found that stimulation of the serotonergic dorsal raphe nucleus (DRN) afferents to the nucleus accumbens (NAc) abolishes cocaine reward and promotes anti-depressive-like behaviors. Lastly, using a rat model of compulsive-like cocaine self-administration, we found that inhibition of dorsal raphe 5-HT1Aautoreceptors attenuates cocaine self-administration in rats with 6h extended access, but not 1 hour access to the drug. Therefore, our findings suggest an important role for 5-HT1A autoreceptors, and thus DRN→NAc 5-HT neuronal activity, in the etiology and vulnerability to cocaine reward and addiction. Moreover, our findings support a strategy for antagonizing 5-HT1A autoreceptors for treating cocaine addiction.

Review on using virally-mediated DREADD expression

During the past few years, CNO-sensitive designer G protein-coupled receptors (GPCRs) known as DREADDs (designer receptors exclusively activated by designer drugs) have emerged as powerful new tools for the study of GPCR physiology. In this chapter, we present protocols employing adeno-associated viruses (AAVs) to express a Gq-coupled DREADD (Dq) in two metabolically important cell types, AgRP neurons of the hypothalamus and hepatocytes of the liver. We also provide examples dealing with the metabolic analysis of the Dq mutant mice after administration of CNO in vivo. The approaches described in this chapter can be applied to other members of the DREADD family and, of course, different cell types. It is likely that the use of DREADD technology will identify physiologically important signaling pathways that can be targeted for therapeutic purposes.

Nice video of glial DREADD activation in enteric nervous system.

I get occasional questions regarding use of DREADDs outside the CNS and in non-neurons.  Here's a paper where glial localized hM3Dq was activated.  The video is pretty cool as well.

DREADDs to decostruct visceral pain

"The activity of neurons in pancreatitis-related pain centers was pharmacogenetically modulated by DREADDs, selectively and cell type-specifically expressed in target neurons using AAV-mediated gene transfer. Pharmacogenetic inhibition of PVT, but not PAG neurons attenuated visceral pain, and induced an activation of the descending inhibitory pain pathway. Activation of glutamatergic principle neurons in the mPFC, but not inhibitory neurons, also reversed visceral nociception."

Abbreviations:  the paraventricular nucleus of the thalamus (PVT), the periaqueductal grey (PAG)

CNO via osmotic minipump

I get occasional questions about this and have not tried this in my lab but was alerted to an on-line thesis which has successfully used this approach for therapeutic purposes in Alzheimer's Disease models in mice.

Typical combination opto- chemogenetic paper--this time odorant system

I'm frequently asked

"Which is better, opto- or chemogenetics?"

To which I'm tempted to reply:  "Which is better, red or green?"

Clearly, depends on the experiment and it is likely that there is a perfect (or nearly so) way to do it.  Sometimes this will be with light, other times with chemicals--sometimes neither.

At any rate, here's a nice example of combining them to deconstruct interneuron activity and odorant in Nature Neurosciences

"Neuronal pattern separation is thought to enable the brain to disambiguate sensory stimuli with overlapping features, thereby extracting valuable information. In the olfactory system, it remains unknown whether pattern separation acts as a driving force for sensory discrimination and the learning thereof. We found that overlapping odor-evoked input patterns to the mouse olfactory bulb (OB) were dynamically reformatted in the network on the timescale of a single breath, giving rise to separated patterns of activity…

Identifying Dorsal Horn Circuits for Persistent Mechanical Pain with DREADDs

Nice paper here in Neuron today as well as commentary.

One of interesting tidbits (for me at least) was that apparently DREADD technology is so widely used that the authors no longer cite any papers (or reviews) to it as a technology--I guess this means anyone will know what they mean....

DREADDs in Lentiviral vectors

For reasons that are not entirely clear to me we are getting many requests for hM4Di and hM3Dq in lentiviral vectors.

We will make what we have available via ADDGENE over the next couple of weeks.

Effect of KORD on palatable food ingestion

A nice paper on the effect of food palatability and feeding as well as a nice independent replication of effects of KORD in Cell Metabolism

Using DREADDS to deconstruct olfaction

Interesting paper here:  "the chemogenetic reduction of cholinergic activity in freely behaving animals disrupted odor discrimination of simple odors, and the investigation of social odors associated with behaviors signaled by the Vomeronasal system."

Chemogenetic Inactivation of Dorsal Anterior Cingulate Cortex Neurons Disrupts Attentional Behavior in Mouse

"We virally expressed inhibitory hM4Di designer receptor exclusively activated by a designer drug (DREADD) in dACC neurons, and examined the effects of this inhibitory action with the attention-based 5-choice serial reaction time task. DREADD inactivation of the dACC neurons during the task significantly increased omission and correct response latencies, indicating that the neuronal activities of dACC contribute to attention and processing speed. Selective inactivation of excitatory neurons in the dACC not only increased omission, but also decreased accuracy. The effect of inactivating dACC neurons was selective to attention as response control, motivation and locomotion remain normal. This finding suggests that dACC excitatory neurons play a principal role in modulating attention to task relevant stimuli. This study establishes a foundation to chemogenetically dissect specific cell-type and circuit mechanisms underlying attentional behaviors in a genetically tractable species.&q…

Here's another in vivo hM4Di paper

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Nature, Trachtenberg lab GCaMP6-expressing PV neurons in vivo.

and a nice image from a Neuron paper demonstrating same


In vivo evidence for suppression of neuronal firing by hM4Di

I received an email last week asking if there was any evidence of this (other than readouts like cFos and bath application of CNO to slices).

There is a nice paper in Nature 2015 (Fig 4) using GCAMP as  a readout, here in Nature Communications using power-spectrum EEG as  a readout and in Neuron using single unit recordings in vivo.

There are >100 papers citing use of hM4Di for this purpose in vivo but most (not surprisingly) look at slices to validate silencing/suppression of activity.

Reversible Inactivation of the Dorsolateral Prefrontal Cortex in Non- Human Primates via Designer Receptors Exclusively Activated by Designer Drugs Impairs a Spatial Memory Task

Interesting preliminary data reported in a published thesis on-line.  Nice control data and promising enough.

Dual virus system for targeted expression of opto- and chemogenetic cargos via AAV

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Interesting and useful approach out now as outlined in figure below (from paper)




"Ultimately a library of subtype specific Cre viruses could be created, fully characterized in specific brain regions/species, and made available to the research community."

Retro-DREADDs for pathway mapping and modulation in primates

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A very nice 'proof-of-concept' study to show feasibility of using Retro-DREADD technology in primates as shown in Figure 1 from paper


"The aim of the inclusion of DREADDs in the AAV5 is to test the feasibility of DREADD expression on the specific prefrontal neurons in the macaque brain for future pharmacogenetic manipulation studies. Although we did not test the behavioral and neuronal effects of CNO administration in this study, the observation of mCherry-positive neurons highly likely indicates DREADD expression in our double infection system. "

DREADDs to deconstruct role of GLP-1 and feeding

"Using chemogenetic tools, we discovered that central GLP-1 acts on the midbrain ventral tegmental area (VTA) and suppresses high-fat food intake. We used integrated pathway tracing and synaptic physiology to further demonstrate that activation of GLP-1 receptors specifically reduces the excitatory synaptic strength of dopamine (DA) neurons within the VTA that project to the nucleus accumbens (NAc) medial shell. These data suggest that GLP-1 released from NTS neurons can reduce highly palatable food intake by suppressing mesolimbic DA signaling."

New Video on DREADDS; tutorial

DREADDs, neurotensin and DA efflux

DREADDs regulating reward

DREADDs to interrogate circuits in brain injury recovery

DREADDs and deconstructing circuits involved in decision making

Role for insular system here.

DREADDs in rats: regulation of LC neurons via TH-transgenic rats to control appetitive responses

Nice paper here

"Using designer receptors exclusively activated by designer drugs (DREADD) in transgenic rats expressing Cre recombinase under the control of tyrosine hydroxylase promoter, catecholamine neurons in cell groups A1 and C1 of the ventrolateral medulla were activated selectively by peripheral injection of clozapine-N-oxide (CNO). CNO injection increased food intake and c-Fos expression in PeFLH orexin neurons, as well as in PVH neurons. In summary, catecholamine neurons are required for activation of orexin neurons during glucoprivation. Activation of orexin neurons may contribute to appetitive responses required for glucoprivic feeding."

DREADDs and dimerization

Yet another paper showing no evidence for DREADD-mediated hetereodimerization of GPCRs (at least in transfected cells) being of any biochemical significance.

Using DREADDS + Optogenetics to deconstruct co-transmission (Histamine and GABA) for wakefulness

A nicely elegant study--much here to ponder.

Interesting report of DREADDs outside the CNS in non- neuronal tissues

Nicely informative (and short) video introducing DREADD technology

Found here

KOR-DREADD in rats

The first independent replication of neuronal silencing and behavioral modulation by KOR-DREADD (KORD) has beenpublished.

DREADDs + Opto to deconstruct circuits in learning and memory

Nice combined use of both technologies to interrogate memory and learning .

KOR DREADD now truly available via ADDGENE

After some delay, the link for KOR DREADD is now active at ADDGENE.

Using DREADDs to interrogate signaling in striatum as it relates to behavior

ADDGENE links to new DREADDs

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A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior

Now on-line at Neuron.

The constructs will be available soon from ADDGENE  without MTA.

Viruses will be available next week (May 3) from UNC.

This DREADD is activated by salvinorin B which may be purchased from Cayman or from Scott Wyer at Apple Pharms at a great discount.

Using DREADDs to identify cells involved in mediating aggression

Interesting paper in Cell Reportson this topic.

Using DREADDs to deconstruct contribution of astrocytes to feeding

PRESTO-TANGO: An open-source GPCR-ome resource for validating selectivity of chemogenetic actuators

Here is a paper just published which provides for an open-source resource for screening the druggable human GPCR-ome for off-target actions of chemogenetic chemical actuators.  The cDNAs will soon be available from ADDGENE and are available until then from the Roth lab.

Tet-tagged DREADDs

Using DREADDs to deconstruct pathways essential for treating neuropsychiatric disease

Nice paper in PNAS by Caron's lab.

DREADDs vs OPTO: does it matter to the neuron?

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I get this question a lot and certainly there are a number of distinctions one can make with regard to these two technologies (Table 1).

TABLE 1. COMPARISON OF CHEMO- AND OPTOGENETIC PLATFORMS