Optogenetic investigation of neural circuits underlying brain disease in animal models

Abstract

Optogenetic tools have provided a new way to establish causal relationships between brain activity and behaviour in health and disease. Although no animal model captures human disease precisely, behaviours that recapitulate disease symptoms may be elicited and modulated by optogenetic methods, including behaviours that are relevant to anxiety, fear, depression, addiction, autism and parkinsonism. The rapid proliferation of optogenetic reagents together with the swift advancement of strategies for implementation has created new opportunities for causal and precise dissection of the circuits underlying brain diseases in animal models.

Figure 1 |. Optogenetic tools.

a | Major classes of single-component optogenetic tools include cation-permeable channels for membrane depolarization (such as channelrhodopsins (ChRs)), chloride pumps (for example, halorhodopsin (NpHR)) and proton pumps (such as bacteriorhodopsin or proteorhodopsin (BR/PR)) for membrane hyperpolarization, and light-activated membrane-bound G protein-coupled (OptoXR) or soluble (bacterial cyclase) receptors that mimic various signalling cascades. b | Tools that have been characterized in terms of wavelength activation spectra and decay kinetics. The chart shows peak activation wavelength plotted against decay kinetics and illustrates groupings of tools over the range of spectral and temporal characteristics. This also demonstrates why it is feasible to use tools that are well separated in spectral and/or temporal domains to achieve dual-channel control. It should be noted that the kinetics for OptoXR were characterized in vivo using an assay that measured a downstream readout (spiking) and probably represent an upper bound for these properties. Decay kinetics are temperature-dependent; all reported values except for channelrhodopsin-green receiver (ChRGR) were recorded at room temperature (an ~50% decrease in decay kinetics is expected if the temperature is increased to 37°C). ChRGR has only been studied at 34°C. We have therefore extrapolated the likely range for this protein at room temperature. The decay kinetics for the L132C mutation (calcium translocating channelrhodopsin (CatCH)) were not measured in neurons, and these properties may depend on factors including the presence of other channels in the host cell and the host cell tolerance of, and response to, elevated intracellular Ca²⁺ levels. The recently determined crystal structure of ChR2 may allow the design of additional classes of optogenetic tools. The ‘/’ indicates the combination of two mutations. Figure is modified, with permission, from REF. © (2011) Elsevier. Arch, archaerhodopsin; bPAC, bacterial photoactivated adenylyl cyclase; C1V1, ChR1/VChR1 chimaera; cAMP, cyclic AMP; DAG, diacylglycerol; eBR, enhanced bacteriorhodopsin; InsP₃, inositol trisphosphate; VChR1, Volvox channelrhodopsin 1.

Figure 2 |. Targeting strategies with optogenetic tools in vivo.

a | Neuronal cell bodies can be directly stimulated by injecting a viral vector into the target region and implanting a local light-delivery device in the same region. b | Specific expression of the transgene in defined cell populations can be achieved by including cell-type-specific promoters within the viral vector or by injecting a recombinase-dependent virus into an animal that is engineered to express a recombinase (such as Cre) in particular cell types. c | The optogenetic tool can be targeted to axonal projections by injecting the virus at the location of neuronal cell bodies and delivering light to the target region harbouring opsin-expressing processes. d | In projection termination labelling, cells are targeted by virtue of their synaptic connectivity to the target region, probably excluding axons that are simply passing through the area. In the example shown, transcellular labelling is achieved using a recombinase-dependent system. The synaptic target site is injected with a virus expressing Cre that is fused to a transneuronal tracer (such as a lectin), and the cell body region is injected with a Cre-dependent virus. This results in cells that project to the Cre-injected area becoming light sensitive. Similar effects can be obtained using retrograde viruses (those that transduce the axon terminal), such as rabies or herpes simplex viruses (HSVs), although these approaches do not enable control over the postsynaptic cell type. e,f | Combinatorial manipulations at either neuronal somata (e) or projections (f) can be achieved with two different optogenetic tools that have well-separated activation spectra (responding to different wavelengths of light) and by using a light-delivery tool to merge multiple wavelengths of light. Figure is modified, with permission, from REF. © (2011) Elsevier.

Figure 3 |. Functional dissection of amygdala microcircuitry using an integrated approach involving optogenetic tools.

a–d | Four recent papers^,,, have used optogenetic tools to dissect subpopulations of amygdala neurons or projections in fear and anxiety studies. The diagrams show connectivity but are not intended to depict ultrastructural anatomy. a | In one study, adeno-associated virus (AAV) 2/1 serotype was used to express channelrhodopsin 2 (ChR2) in glutamate neurons in the lateral amygdala (LA), and LA somata illumination (in lieu of shock) was paired with a conditioned stimulus to produce fear responses. This showed that activation of LA neurons produced unconditioned fear responses (freezing), which when paired with a stimulus was able to support stimulus-evoked freezing. b,c | Recent studies^, identified two interesting subpopulations of neurons in the centrolateral nucleus of the amygdala (CeL). In the experiment shown in b, a recombinant AAV (rAAV) was used to express ChR2 in the CeL and illumination of the centromedial nucleus of the amygdala (CeM) was used to produce fear responses (b). When the conditioned stimulus was presented, two populations of CeL neurons were identified: CeL On cells showed excitation, whereas CeL Off cells were inhibited. In the experiment shown in c, AAV2/5 expressing ChR2 was injected into the CeL of protein kinase Cδ (PKCδ)::GluClα-ires–Cre mice to show that PKCδ-expressing neurons inhibited periaqueductal grey (PAG)-projecting CeM neurons (c). PKCδ⁺ cells corresponded to CeL Off cells. d | Another study examined amygdala function in the context of unconditioned anxiety rather than conditioned fear. In this study, basolateral amygdala (BLA) neurons were transduced with an AAV expressing ChR2, and it was shown that activating BLA–CeL projections reduced anxiety-related behaviours, whereas activating the BLA cell bodies without specificity for projection target increased anxiety-related behaviours. This study also demonstrated the opposite effects of BLA–CeL projections using optogenetic inhibition. However, this study did not determine whether BLA neurons provided monosynaptic excitatory input to a particular subpopulation of CeL neurons. e | Information from these four studies, synthesized to display certain current optogenetically obtained knowledge about amygdala microcircuitry that is causally involved in behaviour. Although many questions remain, these studies show the advantages of integrating optogenetic tools with molecular, electrophysiological, pharmacological and imaging techniques in the context of behaviour relevant to psychiatric disease.

Figure 4 |. Optogenetic dissection of limbic circuits in the context of reward-seeking behaviour.

A schematic diagram (top panel), showing key neural projections involved in reward, and an expanded view of intra-accumbens microcircuitry (bottom panel; numbers indicate independent findings). The diagram shows connectivity but is not intended to depict ultrastructural anatomy. Cholinergic interneurons expressing choline acetyltransferase (ChAT) in the nucleus accumbens (NAc) modulate the activity of medium spiny neurons (MSNs) and modulate the ability of the animal to develop cocaine-conditioned place preference (1). Mice will readily work (nose poke) to receive illumination of channelrhodopsin 2 (ChR2)-expressing basolateral amygdala (BLA) axon terminals in the NAc (2). This self-stimulation behaviour is D1 dopamine receptor (D1R)-dependent, and does not occur when ChR2-expressing prefrontal cortex (PFC) axon terminals in the NAc are illuminated. It has been shown that activation of dopaminergic neurons in the ventral tegmental area (VTA) can support operant responding in both rats and mice, and in rats, illumination of tyrosine hydroxylase and ChR2-expressing axon terminals in the NAc also supports operant responding (3). The activation of D1R- or D2R-expressing NAc neurons shows differential effects on cocaine-conditioned place preference (4).

Figure 5 |. Functional mapping of basal ganglia circuitry using optogenetics in the context of Parkinson’s disease.

A schematic diagram (top panel) shows key neural projections that are involved in parkinsonian behaviour and treatment. Data in the bottom left panel are from a study that used a constitutively expressing channelrhodopsin 2 (ChR2) mouse line (Thy1::ChR2) to identify a mechanistic explanation for the therapeutic effects of deep brain stimulation (DBS). By illuminating and recording in the subthalamic nucleus (STN), this paper showed that afferent fibres entering the STN, rather than local cell bodies themselves, are likely to be the direct target of DBS in the correction of parkinsonian motor activity. High-frequency stimulation (HFS) of the afferent fibres into STN potently silenced the structure as shown and reversibly abolished the parkinsonian symptoms. By contrast, low-frequency stimulation (LFS) of the afferents simply added spikes on top of endogenous spikes and worsened parkinsonian symptoms. Data in the bottom right panel are from a study that used a Cre-dependent adeno-associated virus (AAV) to selectively express ChR2 in either D1 dopamine receptor (D1R)::Cre or D2R::Cre mice to examine the differential contributions of the direct and indirect pathways with respect to motor output. Activation of D1R-expressing neurons silenced local basal ganglia activity and increased ambulation, whereas activation of D2R-expressing neurons increased this activity and enhanced immobile or bradykinetic (slow) behaviour. Black bars indicate the duration of illumination. The bottom left panel is reproduced, with permission, from REF. © (2009) American Association for the Advancement of Science. The bottom right panel is reproduced, with permission, from REF. © (2010) Macmillan Publishers Ltd. All rights reserved. GP, globus pallidus; M1, primary motor cortex; SNr, substantia nigra pars reticulata.