|Fig. 1: Steps involved in projection targeting, which allows anatomically-targeted optogenetic manipulation. For example, if one wishes to
target only the pathway from B to A, illumination at the same locus as virus injection (B) would be counterproductivesince C is affected.
Transfected axons can be illuminated directly at their terminals (A), allowing for simple, anatomy based targeting. Modified from .
Optogenetics has grown to be applied to nearly every subfield of neuroscience. As new lines of investigation are launched, optogenetic tools by themselves are no longer adequate, and need to be altered and used in new approaches. Opsins have been altered in different ways: they have been refined for better performance (ChETA); redshifted to allow different wavelengths of excitation (C1V1); slowed down to facilitate longer, subthreshold depolarizations; and new opsin classes continue to be investigated as tools for manipulating intracellular signaling (OptoXR). Optogenetics also continues to be applied in novel, creative experimental strategies: techniques such as projection targeting can achieve excellent anatomical specificity, and the integration of optogenetics with existing transgenic techniques has proven to be a powerful tool for targeting neurons based on their intrinsic biochemistries. These new tools and techniques have been applied in a number of ways, allowing for the unprecedented deconstruction of emergent and complex behavioural and pathological phenomena.
1 Temporal specificity: slow kinetics and bistability
As an optogenetic paradigm, the utility of bistable, slow opsins is not immediately obvious compared to opsins with fast kinetics. However, as a greater role for intrinsic plasticity was realized in the modulation of behaviour, likewise, a greater need for such tools was also realized. Slow kinetics refers to the slow deactivation time for a channel after a light pulse, allowing current to pass for a far longer amount of time than physiologically typical for a channel, whereas bistable means that the opsin can be activated and deactivated with different and distinct wavelengths of light. Bistable opsins with slow kinetics can allow for manipulation of neuronal intrinsic excitability on the timescale of tens of minutes, along with the ability to remove the fiber optic from the animal during subsequent behavioural testing, reducing any sensory/motor impairments associated with a tethering apparatus . Bistable opsins with slow kinetics have been termed “step-function opsins” (or SFOs) for their step-function-like ability to elevate neuronal excitability. These tools allow for avenues of scientific inquiry not previously possible with traditional electrical or lesion based techniques.
1.1 Autism and schizophrenia
|Fig. 2: Behavioural and physiological disease correlates elicited by increased
E/I balance in mouse mPFC. a) Deficits in social behaviour and fear memory learning
are observed in animals with increased, but not decreased, E/I ratios. Deficits were
rescued by allowing for eventual inactivation of opsins. b) Social deficits are observed
in animals with increased E/I ratios localized to the PFC; increased E/I in V1 showed
normal social behaviour. c) High frequency activity in the 30-80 Hz range, elicited by
photoactivation (473nm) of SSFOs and rescued by photodeactivation (594nm). Modified
A long-standing, yet hard-to-evaluate theory behind psychiatric disorders (autism and schizophrenia, in particular) was the neuronal excitation/inhibition (E/I) imbalance hypothesis. As these disorders arise from a multitude of distinct genetic and environmental causes, the validation of such a hypothesis can unify these diverse etiologies into a well-defined physiological target for therapeutic intervention. Difficulty in evaluating this hypothesis is in part due to the fact that psychiatric disorders often involve an interplay of different intrinsic and extrinsic factors, making it difficult to artificially produce an emergent pathological phenotype; for example, the impact of genetic manipulations altering neuronal excitability may be diminished by compensatory processes that can restore near-normal function. Yizhar et al. were able to acutely manipulate the E/I balance in transgenic animals using stabilized step-function opsins (SSFOs), thus avoiding problems due to functional compensations, and tested the E/I imbalance hypothesis directly in this way. Elevation of the E/I balance in the mouse medial prefrontal cortex (mPFC) resulted in various behavioural and physiological disease-state correlates, including deficits in social behaviour, deficits in fear memory learning (figure 2a), and high frequency (30-80Hz) baseline activity (figure 2c) . Other manipulations such as reducing E/I balance (via coupling of SSFO with the parvalbumin promoter) or elevation of E/I balance in other cortical regions (specifically, the primary visual cortex; V1) did not produce the same effects (figure 2b), suggesting a cortical and physiological specificity. Yizhar et al. also tested the effects of increasing inhibition to rescue an elevated E/I balance by using combinatorial optogenetics, in which both excitatory and inhibitory neurons expressed either SSFO or a redshifted channelrhodopsin variant (C1V1), respectively. While social deficits were not completely ameliorated, rats expressing both CaMKII::SSFO and PV::C1V1 had a slight but significant recovery of social behaviour preference after treatment . Taken together, this work lends support to the E/I imbalance hypothesis, provides a novel, optogenetic animal model for disorders such as autism and schizophrenia, and demonstrates the value of optogenetics in evaluating previously untestable hypotheses.
2 Techniques for spatial and histological specificity
|Fig. 3: Experimental setup and results. a) Diagrams of the automated FST
setup and projection targeting strategy. b) Photostimulation of mPFC neurons in the
mPFC was ineffective at eliciting an antidepressive response, but projection targeting
of the mPFC-DRN pathway produced rapid, profound, stimulation dependent effects.
Modified from .
Optogenetics can target specific brain regions, or even specific cells, in serveral ways. Most obviously, injection of virus containing an opsin gene can be targeted using stereotaxic surgery. Even at this basic level of targeting, optogenetics already confers specificity advantages over electrical stimulation, as the virus will not effectively transfect passing axons. Thus, transfection is localized to present neuronal soma only, and optical stimulation of the same locus will only excite these neurons and not those with axons passing by. Several other methods have been leveraged to induce cell specific expression of opsins. An interesting strategy, unique to optogenetics, is “projection targeting”, whereby virus is injected into one locus but illumination occurs at another. Expressed opsins are shuttled effectively down neuronal processes, so transfection at one anatomical locus can result in opsin expression in distant loci along axonal projections. This way, with proper knowledge of anatomical features in target regions, one can exactingly target cell populations defined by their anatomical projections. Combination of optogenetics with existing transgenic techniques (such as the Cre-Lox system) is another strategy, particularly useful for the ability to couple opsin expression with the intrinsic biochemical features of specific neuronal subtypes. By coupling opsin or Cre recombinase sequences to neuronal-subtype-specific promoters, one can drive opsin expression in inhibitory neurons only or serotonergic neurons only, for example. The multitude of possible targeting strategies makes optogenetics a versatile and powerful investigational tool.
There are a number of treatments for depressive disorders in humans; the most well-known are a class of drugs called selective serotonin reuptake inhibitors (SSRIs). While these drugs have been effective in a great number of patients, a major weakness is their delayed onset of action; patients typically begin to report positive results only after weeks or even months after treatment begins. This delay can be fatal in depressed, suicidal patients. One promising therapy, known as deep brain stimulation (DBS), has been shown to safely and rapidly elevate moods in depressed individuals. The therapy involves electrical stimulation of the subgenual (SG) cortex of the prefrontal cortex (PFC), a region heavily involved in executive function with projections to many brain structures. While DBS is effective, its mechanisms of action are unclear – PFC projections are ubiquitous to many brain regions and heterogenous in their effects.
To investigate possible neural projections involved in stress and behavioural challenge, Warden et al. transfected rat excitatory medial PFC (mPFC; a rodent homologue of the SG) cells with channelrhodopsin-2 (ChR2) under control of the CaMKIIα promoter. However, subsequent optical stimulation was surprisingly ineffective (figure 3b) at eliciting less immobility in the forced swim test (FST), an animal behavioural assay for learned helplessness and depression (figure 3a). While surprising, this result should perhaps be expected given the heterogenous nature of mPFC projections; different projections might elicit opposing effects, resulting in no net change in behavior. Further work targeted the Dorsal Raphe Nucleus (DRN), seeing as the DRN is a major serotonergic nucleus and has been implicated in depression. A projection targeting strategy (figure 1, 3a) was employed to selectively excite only the mPFC-DRN pathway. To do this, virus was again injected into the mPFC; however, optical stimulation was applied only at the DRN, thus exciting only those mPFC axons projecting to the DRN. The result was a profound increase in activity in the FST, and absence of stimulation rapidly reversed antidepressive effects (figure 3b). Thus, the mPFC – DRN projection is involved in the response to stress and behavioural challenge, and its human homologue may be implicated in DBS. This study demonstrates the power of optogenetics to reduce complex networks and behaviours down to single, causal pathways.
|Fig. 4: Experimental setup and results. a) Double-floxed inverted arrangement of ChR2 sequence; when transfected into TH::Cre cells, the inverted ChR2 is un-inverted
and ChR2 transcription can take place. b) Diagram showing site of photostimulation (VTA). c) Heatmap of CPP elicited by phasic dopaminergic stimulation versus tonic stimulation.
d) Graph of CPP elicited by phasic versus tonic stimulation. e) Measurements of dopamine transients in the NAc; phasic stimulation evoked far greater dopamine transients.
Modified from .
Dopamine has long been believed to be involved in the reward and pleasure pathways subserving addiction. Pioneering studies revealed that electrical stimulation of certain brain regions could cause behavioural reinforcement ], and studies involving pharmacological manipulation or lesioning strongly suggested that dopamine is a modulator of motivation and behavioural drive. Despite this, a causal role for dopamine and relevant neuronal circuitry in behavioural conditioning was never conclusively established – electrical stimulation of dopaminergic pathways believed to be involved in reward also resulted in the unavoidable stimulation of nearby tissues and cells. Nearby cell bodies, and any axons passing near the electrode would be inadvertently affected, resulting in unaccountable confounds. Lesion studies also suffered from the same problem of poor spatial specificity.
To address this problem, Tsai et al. used an optogenetic approach coupled with the Cre-Lox system. The gene encoding Cre recombinase was coupled to the tyrosine hydroxylase (TH) promoter in transgenic mice, such that only in cells expressing TH (an enzyme required for the biosynthesis of dopamine) would Cre be expressed. This strategy conferred cell-type specificity for dopaminergic cells only. An inverted channelrhodopsin-2 (ChR2) sequence was then packaged into a virus, flanked by inverted double-floxed (lox2272/loxP) sites and injected into the ventral tegmental area (figure 4a); the VTA is a major dopaminergic nucleus and has been strongly implicated in reward pathways. Although surrounding, non-target tissues and cells might also be transfected by the virus, only those expressing Cre are able to invert the inverted ChR2 sequence into the correct reading frame for transcription – and since only TH cells express Cre in the TH::Cre transgenic mouse, ChR2 expression was effectively targeted to VTA dopaminergic neurons only. Subsequent optical stimulation would then selectively depolarize these dopaminergic neurons exclusively and leave surrounding, non-target tissues unaffected.
Tsai et al. then used behavioural testing to observe the differential effects of dopaminergic firing patterns. Using the conditioned place preference (CPP) paradigm, mice were tested in a chamber with 2 rooms: a “phasic” room where mice were conditioned with 50 Hz phasic photostimulation; a “tonic” room where mice were conditioned with photostimulation at 1 Hz instead. Mice showed a clear preference for phasic stimulation (figure 4c and d), likely due to the greater dopamine transients in the nucleus accumbens (NAc) (a major target of VTA innervation; figure 4e) elicited by phasic stimulation versus tonic stimulation. This study confirms that dopamine as a modulator of behavioural conditioning, that dopaminergic phasic firing is sufficient for behavioural conditioning, and is a demonstration for the spatial specificity and explanatory power afforded by optogenetics coupled with existing transgenic techniques.
2.3 Learning and memory
|Fig. 5: Method for delineating and reactivating a hippocampal engram. a/b) Schematic of setup; ChR2
expression is dependent on the absence of Dox and neuronal activity (driving c-fos and tTA expression).
Neurons activated in the formation of a memory are thus labelled. c) Experimental time course; removal of
Dox from diet facilitates ChR2 expression, allowing for the establishment of a brief time window in which
memory-related neurons can be selectively targeted for ChR2 labeling. d) Optical stimulation of ChR2-labeled
neurons elicits fear memory recall, evidenced by increased freezing response during the light-on epochs;
e) this increase is absent when no shock was applied during fear conditioning (NS); f) and is also absent
without ChR2. Modified from .
One of the most sought after goals of neuroscience research is the ability to delineate the molecules, neurons, or otherwise substrates that constitute a specific long-term memory. To demarcate this hypothetical “memory trace” or “engram” would provide a clear physical representation of a seemingly ethereal mental process. Previous studies have approached this problem using different strategies; for example, Han et al. identified CREB-upregulated neuronal populations in the lateral amygdala that, when selectively ablated, caused disruption of a previously learned fear memory . While such studies have been instrumental in discovering the neural substrates of learning, they lack positive evidence for the presence of a memory, instead relying on memory disruption. Liu et al. used an optogenetic approach coupled with the tetracycline inducible expression system . In transgenic animals, tetracycline transactivator (tTa) was coupled to the promoter of c-fos, an immediate early gene whose expression signals recent cell activation (figure 5a and b). Dentate gyrus (DG) granule cells of the hippocampus were transfected with viruses containing channelrhodopsin-2 (ChR2) downstream of the tetracycline response element (TRE). Thus, if DG cells were activated, tTa would be expressed, driving the expression of ChR2. To prevent ubiquitous ChR2 expression in response to non-specific DG activity, rats were fed doxycycline (Dox), an inhibitor of tTa and thus prevents ChR2 expression. During fear conditioning, rats were taken off Dox, allowing for ChR2 expression in response to fear memory learning (figure 5c). Neurons activated during this learning would express c-fos, and by extension, ChR2, resulting in a labeling of the specific neurons constituting a memory. Later, in a different context, DG cells were optically stimulated; this triggered freezing responses (figure 5d) indicating that i) activated neurons represented a memory trace, and ii) activation of these neurons was enough for memory recall. Here, the marriage of optogenetics to existing genetic techniques provides an impressive novel tool for not only delineating, but also recalling an engram.