5a. Methods for Inactivation
Reversible inactivation of genetically targeted neuronal populations has been achieved either by blocking synaptic transmission or by abolishing action potential generation. shibirets
, a dominant temperature-sensitive mutation of Drosophila
dynamin, is the prototypical inducible method for inactivation. In neurons expressing shibirets
, inactivation is triggered by raising the temperature from room temperature to ~30°C. At elevated temperatures endocytosis of synaptic vesicles grinds to a halt, leading to the depletion of the synaptic vesicle pool and rundown of synaptic transmission. Induction and reversal occurs within a few minutes after the temperature shift (Koenig et al., 1983
; Kitamoto, 2001
). In Drosophila
has been used to dissect the circuits underlying memory formation, courtship behavior, and olfactory processing (Waddell et al., 2000
; Dubnau et al., 2001
; McGuire et al., 2001
; Manoli et al., 2005
; Stockinger et al., 2005
). One concern with shibirets
is that dynamin has many roles in membrane trafficking that extend beyond synaptic transmission. In addition, temperature shift as an inducer is not generalizable to higher vertebrates.
Many proteins in synaptic terminals are specific and essential for synaptic transmission (reviewed in Fernandez-Chacon and Sudhof, 1999
). The intricately choreographed sequence of protein-protein interactions leading to vesicle fusion and vesicle recycling provides numerous potential protein targets for inducible inactivation. Inducible expression of tetanus toxin light chain (TeTxLc) has been used to inactivate synaptic transmission in flies (Sweeney et al., 1995
) and mice (Yamamoto et al., 2003
). But the time course of induction and reversal are slow ().
More recently, protein crosslinking induced by small-molecule dimerizers (Spencer et al., 1993
) was used to develop Molecular Systems for Inactivation of Synaptic Transmission (MISTs) (Karpova et al., 2005
). MISTs consist of modified synaptic proteins that can be crosslinked by the addition of small molecule dimerizers to block aspects of the synaptic vesicle cycle. In excitatory (Karpova et al., 2005
) and inhibitory (D. Tervo, T. Sudhof, K.S., A. Karpova, unpublished data) MIST-positive neurons in vitro, application of dimerizer induces inactivation of synaptic transmission within tens of minutes. Reversal occurs over 1 hr. When targeted to Purkinje cells in transgenic mice, MISTs interfere inducibly and reversibly with performance in a cerebellum-dependent behavioral assay (Karpova et al., 2005
MISTs have three drawbacks. First, it is not clear if dimerizers cross the blood-brain barrier (BBB), and in vivo applications so far have relied on intracerebroventricular (ICV) delivery. Second, in the wild-type background, MISTs compete with endogenous synaptic proteins; as a result, efficient inactivation of synaptic transmission requires high-level expression of the transgenes. Third, assessing the efficacy of MIST-dependent silencing in vivo can be challenging since the targets of the MIST-positive cells need to be known.
Other inducible systems silence neurons by manipulating the membrane potential or membrane conductance. The Drosophila
allatostatin (AL) receptor (AlstR) has been expressed in mammalian neurons in vitro and in vivo. Application of the peptide AL opens G protein-coupled inward rectifier K+
channels, counteracting action potential generation. Inducible and rapidly reversible AL-dependent silencing has been demonstrated in vitro (Lechner et al., 2002
) and in a variety of anesthetized animals, including the monkey (Tan et al., 2006
). Induction and reversal is rapid, at ~10 min. The main drawback of the AlstR/AL system is that AL does not cross the BBB. As a consequence, its use in freely moving animals in vivo requires insertion of a catheter for ICV administration (Tan et al., 2008
). Other methods relying on G protein-coupled receptors activated by small molecules could overcome this limitation (Armbruster et al., 2007
Another strategy relying on changing the membrane conductance is based on expression of the ivermectin (IVM)-gated Cl−
channel (GluCl). This system has the advantage of being induced by a compound that can cross the BBB. GluCl consists of α and β subunits which must be coexpressed to form a functional channel. Cultured neurons expressing GluCl can be silenced in an IVM-dependent manner (Slimko et al., 2002
). IVM increases the Cl−
conductance of the membrane, shunting action potential generation. GluCl has been engineered for reduced sensitivity to glutamate (Li et al., 2002
). The GluCl/IVM system has been tested in amphetamine-dependent rotational behavior in mice. In mice expressing GluCl unilaterally in the striatum, systemic administration of IVM caused unidirectional rotation of the animal, indicating that striatal neurons were silenced (Lerchner et al., 2007
The GluCl/IVM system has several potential problems as a silencing strategy. First, because IVM is a glutamate receptor agonist, the effective IVM concentrations that need to be administered are potentially toxic. Second, IVM-dependent silencing is only slowly reversible (over a period that takes approximately days), opening up the possibility of compensatory circuit plasticity. Third, two transgenes need to be expressed in the same neuronal population. The requirement for two transgenes can be advantageous if intersectional methods for targeting cell types are desirable (see Section 2).
Engineering of GABAA
-Rs) has recently been used for cell-type-specific silencing (Wulff et al., 2007
). The GABAA
agonist zolpidem binds to the ubiquitous GABAA
γ2 subunit to prolong the duration of inhibitory currents by allo-steric action. A single amino acid substitution, γ2 I77F, abolishes zolpidem binding while leaving GABA binding unperturbed. As a consequence, γ2 I77F knockin mice are insensitive to zolpidem. Zolpidem sensitivity can be reconstituted in genetically defined subsets of neurons by expression of wild-type γ2 protein. In animals expressing zolpidem-sensitivite GABAA
-Rs in Purkinje cells, performance in the rotarod test is reduced within minutes after systemic administration of zolpidem. The virtues of this technique include the excellent pharmacokinetic properties of zolpidem. In addition, zolpidem is an FDA-approved sleeping aid (Ambien), alleviating concerns with toxicity.
The GABAA-R/zolpidem system has some limitations as a silencing strategy. First, targeting zolpidem-sensitive channels relies on replacement of the endogenous gene, which is practical only in mice. Second, zolpidem likely enhances inhibition without full silencing of the target neuron; this could result in ambiguous results. Third, zolpidem binds the interface of the γ2 and α1 subunits, with reduced affinity for α2 and α3 subunits, and no affinity for α4–α6 subunits. Silencing neurons in regions without α1 expression, such as the amygdala, could thus prove challenging.
So far we have discussed strategies that rely on genetically targeted proteinaceous receptors and small molecule or peptide ligands. These pharmacogenetic strategies are relatively slow: infusion of ligands to spatially extended networks of neurons requires seconds to hours, depending on the route of administration and the properties of the ligand. These times are slower than the dynamics of spike trains underlying many behaviors (which are on the scale of milliseconds). In contrast, light has proven to be an excellent trigger for rapid silencing methods.
Expression of vertebrate rhodopsin 4 in CNS neurons can couple light stimuli to opening of potassium channels, reducing action potential frequency (Li et al., 2005
). Rhodopsin 4 also modulates other G protein-coupled channels, for example voltage-gated calcium channels involved in neurotransmitter release, opening up the possibility of light-gated modulation of short-term synaptic plasticity. However, without additional development the lack of functional specificity of rhodopsin 4 is a major drawback of this approach.
Another exciting recent method relies on expression of Natronomonas pharaonis
halorhodospin (NpHR), a light-gated outward chloride pump. In neurons expressing NpHR, pulses of bright yellow light induce rapid hyperpolarization that can abolish action potential generation (Han and Boyden, 2007
; Zhang et al., 2007
). Individual action potentials in complex spike trains could potentially be eliminated using this method. NpHR expression in muscles and motoneurons in C. elegans
can produce light-gated control of C. elegans
locomotor behavior (Zhang et al., 2007
). Since halorhodopsin is a pump, rather than a channel, high expression levels and light intensities are required for inducible silencing. Furthermore, light delivery to activate deep brain structures could be a limiting factor.
In summary, a diverse arsenal of inducible and reversible LOF systems is available (). Interestingly, each method has distinct properties that may make it especially suitable for particular types of experiments. Two-component systems (GluCl, Sph-StxTm-MIST) are ideal if intersectional methods are desirable to restrict expression to specific cell types. Systems induced by light (rhodopsin 4, halorhodopsin) or locally applied ligands (AlstR) may be preferable if spatially restricted activation is desirable. In contrast, if spatially extended neuronal populations are to be silenced, then rapidly diffusible agonists that can be applied systemically are preferred (GluCl, zolpidem-sensitive GABAA-Rs). Local administration of dimerizer to a particular axonal projection originating in MIST-positive neurons could be used to silence selected synaptic pathways.
5b. Methods for Activation
Ideal methods for activation require excellent temporal control. Expression of ligand-gated channels by themselves is not sufficient because long-lasting activation would ultimately inactivate membrane currents and cause neuronal silencing. In the context of an intact circuit, long-lasting activation could cause runaway excitation or strong feedback inhibition, complicating the interpretation of the responses to activation. For these reasons it is necessary to develop systems with temporal control on the order of milliseconds. Light is the only activation trigger for existing techniques that is sufficiently quick for this purpose.
Similar to LOF methods, the development of GOF methods has relied almost exclusively on bioprospecting. One early attempt relies on reconstituting in mammalian cells the signaling pathway coupling light and depolarization used in Drosophila
photoreceptors (Zemelman et al., 2002
). But this method requires expression of multiple gene products, and the temporal precision of activation is poor.
A related method relies on expression of ligand-gated channels that are mostly expressed in the PNS and whose native ligands are not found at high levels in the brain (Tobin et al., 2002
). Uncaging of the caged ligand then allows activation of genetically targeted cells (Zemelman et al., 2003
). Expression of the ATP-gated P2X2 channel, in the presence of caged ATP, has been used to activate flight control circuits in Drosophila
(Lima and Miesenbock, 2005
). An obstacle to using these methods is that the ligand needs to be injected into the brain. In addition, the channels that have been tested in this context have slow deactivation kinetics (on the scale of approximately seconds).
The development of ChR2 has overcome these problems (Nagel et al., 2003
; Boyden et al., 2005
; Li et al., 2005
; Bi et al., 2006
; Zhang and Oertner, 2006
). The potential impact of ChR2 can hardly be overstated. ChR2 has already allowed fast optical control of genetically targeted neuronal populations in vivo (Nagel et al., 2005
; Schroll et al., 2006
; Arenkiel et al., 2007
; Suh et al., 2007
). When combined with behavioral studies, ChR2 photostimulation allows precise tests of hypotheses about how patterns of action potentials in genetically targeted neurons contribute to behavior. For example, photostimulation of ChR2-positive hypocretin neurons accelerated the waking of sleeping mice (Adamantidis et al., 2007
). Calibrated photostimulation allowed a precise estimate of the number of action potentials in neocortical layer 2/3 neurons required to drive a decision task (Huber et al., 2008
). In addition, ChR2 is already beginning to have an impact on neuroanatomy (Section 3d) and the identification of recorded neurons in vivo (Section 4a).
ChR2 may still have some drawbacks. For example, in some systems, including the CNS of adult flies, retinal may not be present at sufficient concentrations for ChR2 function. In addition, the spatiotemporal currents produced by ChR2 differ from the currents produced by synaptic input. This could be a problem for experiments probing dendritic integration.
These limitations have been addressed using engineered channels in combination with tethered, light-switchable agonists and antagonists (Banghart et al., 2004
; Szobota et al., 2007
). For the purposes of circuit analysis, the most powerful system is the light-based ionotropic glutamate receptor (LiGluR) (Szobota et al., 2007
). A light-switchable agonist (MAG) is covalently tethered to a cysteine that is engineered into the ligand binding domain of a glutamate receptor. Near-UV light switches the MAG isomerization from trans
and leads to agonist binding and channel opening. Green light reverses the isomerization and closes the channel. In neurons expressing LiGluR, light pulses can trigger short, postsynaptic currents that resemble normal synaptic transmission as well as prolonged depolarizations. LiGluR requires that MAG is introduced into the tissue of interest. Since LiGluRs are also activated by endogenous glutamate release, their overexpression may alter normal neuronal and circuit properties. Thus, in an ideal experiment, LiGluR might be used in knockin experiments where the endogenous GluR is replaced by LiGluR.
In summary, a diverse arsenal of rapid GOF systems is available (). Because of its simplicity, for most applications ChR2 is the method of choice. However, other systems, such as P2X2/caged-ATP and LiGluR, may fill important niches, for example by providing access to systems that do not produce retinal or by mimicking synaptic currents.