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. Author manuscript; available in PMC: 2014 Jul 1.
Published in final edited form as: NMR Biomed. 2013 Jan 28;26(7):803–809. doi: 10.1002/nbm.2907

Genetic tools to manipulate MRI contrast

Raag D Airan 1,2, Nan Li 1,3, Assaf A Gilad 1,2,3, Galit Pelled 1,3,*
PMCID: PMC3669659  NIHMSID: NIHMS443907  PMID: 23355411

Abstract

Advances in molecular biology in the early 1970s have revolutionized research strategies for studying complex biological processes, which in turn created a high demand for new means to visualize these dynamic biological changes non-invasively and in real-time. In that respect, magnetic resonance imaging (MRI) technology was a perfect fit, due the versatile possibility to alter the different contrast mechanisms. Genetic manipulations are now being translated to MRI trough the development of reporters and sensors, as well as imaging transgenic and knockout mice. In the past few years, a new molecular biology toolset, namely optogenetics, has emerged, which allows for the manipulation of cellular behavior using light. This technology provides a few particularly attractive features for combination with newly developed MRI techniques for probing in vivo cellular, and in particular neural, processes – specifically the ability to control focal, genetically-defined cellular populations with high temporal resolution using equipment that is magnetically inert and does not interact with radiofrequency pulses. Recent works demonstrate that the combination of optogenetics and functional MRI (fMRI) can provide an appropriate platform to investigate in vivo, at the cellular and molecular levels, the neuronal basis of fMRI signals. In addition, this novel combination of optogenetics with fMRI has the potential to resolve pre-synaptic vs. post-synaptic changes of neuronal activity and changes in the activity of large neuronal networks in the context of plasticity associated with development, learning and pathophysiology.

Keywords: optogenetics, fMRI, cortex, animal models, somatosensory cortex, reporter genes, MRI sensors

The molecular biology revolution

The revolution of molecular biology started in the early 1970s with the development of new technologies for transferring genetic material (e.g. genes) from one organism to another (1). This turned out to be a one of the most valuable tools for studying complex biological processes. Throughout the years, many of these principles were applied for developing research tools that can be used to study a variety of genetic and epigenetic events. This created a high demand for new means to visualize these dynamic genetic changes non-invasively and in real-time. In that sense, magnetic resonance imaging (MRI) technology was a perfect fit, due the versatility in altering the different contrast mechanisms. Genetic manipulations were initially used for altering the NMR signal, using magnetic resonance spectroscopy (2–4). Later on, these manipulations were translated to MRI trough development of reporters (5–15) and sensors (16,17), as well as imaging transgenic and knock out mice (18–20). Recently, imaging of transgenic enzymes that convert substrates with artificially elongated T1 by nuclear hyperpolarization was introduced (21). More about reporter genes for MR can be found on this issue (ref Neeman in this issue).

MRI contrast mechanisms

Keeping in mind that MRI has been widely used to characterize genetic changes, (for example to visualize differences in phenotype in transgenic mice), we will focus here only on genetic changes that were made to influence the MRI contrast, and primarily the proton contrast. In principles, three basic contrast mechanisms can be enhanced using genetic tools: (i) spin lactic, longitudinal (T1) relaxation time, (ii) the transverse (T2/T2*) relaxation time and (iii) magnetization transfer / chemical exchange saturation transfer (CEST).

The longitudinal (T1) relaxation of the water protons varies between tissues and can be modified (usually shortened) in the presence of a given “agent” (22,23). This will lead to signal enhancement or brightening of the MR image. In general, genetic alternations that affect T1 contrast are associated with binding of paramagnetic ions or complexes, or changes in the water content of the tissue.

On the other hand, changes in the transverse relaxation (T2) time can either enhance the contrast or reduce it. Form the genetic point of view, T2 relaxation is mostly manipulated by the accumulation of iron (Fe), or changes in the iron oxidation states from diamagnetic to paramagnetic. This was used for certain reporter genes such as ferritin (7–9,19,24,25), the Transferrin receptor (26), melanin (27) and MagA (28).

Genetic manipulations can also be used to indirectly affect blood oxygenation level dependent (BOLD) contrast. In addition to changes in blood volume and blood flow associated with neuronal function, the BOLD contrast is sensitive to changes in the ratio between the oxygenated hemoglobin (which is diamagnetic) and deoxygenated hemoglobin (which is paramagnetic). Therefore, hemoglobin reduction will accelerate MR transverse relaxation (T2 and T2*) due to susceptibility effect. T2 and T2* shortening would reduce the MR signal intensity and consequently enhance the MR contrast (29). BOLD MRI, also known as functional MRI (fMRI), is primarily used for measuring brain activity in human and in small animal models (30). It has been also applied for measuring tumor oxygenation (31) and formation of reactive oxygen species in cancer treatment (32).

CEST contrast, which relies on the proton exchange of the probe or protein with the protons of the surrounding water, reduces the signal intensity and has been used to detect genetically encoded artificial proteins (10) as well as enzymes (15).

Optogenetics for MR physicists: how does it work?

Optogenetics is a new toolset in the molecular biology toolbox that allows for manipulation of cellular behavior using light. As we describe below, this field provides a few particularly attractive features for combination with newly developed MRI techniques for probing in vivo cellular, and in particular neural, processes – namely the ability to control focal, genetically-defined cellular populations with high temporal resolution using equipment that is magnetically inert and does not interact with radiofrequency pulses.

The first major optogenetic protein used to study cellular physiology, Channelrhodopsin-2 (ChR2), takes principally blue light and transduces that light energy into opening its cation channel, thereby inducing cellular depolarization. This protein can be used to make neurons generate an action potential in response to a pulse of blue light, allowing direct control of neuronal action on the millisecond timescale most relevant for neural computation (33). ChR2 forms the prototype of optogenetic molecular devices as it:

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    Transduces applied light energy to allow control of a specific cellular action;

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    Is expressed as a single genetic element, allowing selection of genetically-specified cellular populations for optical manipulation through the use of molecular genetic tools; and

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    Does not require the application of exogenous chemical cofactors or reagents, other than the one-time introduction of the gene encoding the optogenetic device to the cell of interest.

Since the development and publication of ChR2, numerous variant optogenetic devices have been developed, with in-depth reviews of their individual characteristics available (34). Here, we focus on the key features of optogenetic devices and their applications that are useful in combination with MRI experiments (35). In general, the experimental design strategy under consideration will need to take into account the body/brain region and cell-type of interest, as well as, of course, the scientific question under consideration. It is important to keep in mind that the use of optogenetic devices requires the delivery of light to the tissue in question, which generally requires the implantation of optical hardware directed towards the region of interest.

The first choice a researcher interested in using an optogenetics approach for their experiment must make is: which cellular action do I want to control? The principal options currently available are membrane depolarization, e.g. to activate neurons, the exemplar of which is ChR2; membrane hyperpolarization, e.g. to silence neurons, the exemplar of which is Halorhodopsin (NpHR) (36); and modulation of intracellular biochemical signaling pathways, e.g. to mimic the action of hormones or neuromodulators on cells, the exemplars of which are the optoXRs (37). Related questions are: what wavelength of light do I want to use? And what kinetics of activation do I desire? Optogenetic molecular devices generally utilize endogenous retinoids to sense light and have peaks in their input action spectrum principally in the visible light wavelengths, ranging from far red to the far blue (34). Similarly, optogenetic devices have been engineered with a range of kinetics of on and off activation, with the generally more sensitive opsins requiring longer kinetic timescales (38). For most MRI applications, the most well characterized opsin in each family (e.g. ChR2 for neuronal activation) is generally sufficient.

The second major question to be answered is: which cell type do I want to control? The genetics of optogenetics refers to the ability to utilize these molecular devices to isolate genetically specified cellular populations for targeted control. This is usually achieved through placing the gene encoding the opsin under the control of a genetic promoter specific to the cell-type of interest; or, through cell-type specific tropism of the gene delivery method (39). As neural tissue is histologically heterogenous, being able to isolate, for instance, only the excitatory neurons of the cortex by using the CaMKIIα promoter (36), provides a distinct advantage for optogenetic stimulation in comparison to electrical stimulation, which would generally recruit excitatory as well as inhibitory cell types and surrounding glia. Similarly, optogenetic strategies have utilized similar methods to isolate control over hypocretinergic cells (40), dopaminergic cells (41), and parvalbumin-positive interneurons (42). The generation of viral vectors encoding optogenetic devices under the control of a recombinase dependent expression cassette allows for the utilization of Cre-driver mouse lines available for targeting myriad cell types (34). An additional strategy for isolating specific cell-types and inputs is to utilize the ability of neurons to transport these membrane proteins down their far projecting axons. Then, after delivery of the gene to the neuronal cell bodies in one brain region, by delivering light to a downstream brain region, only the synaptic inputs from the initial brain region to the downstream region will be recruited. Further details of targeting strategies are discussed in available technical reviews (34).

The third question to answer in designing an optogenetic experiment is: where is my region of interest and how should I deliver light there? The ability to focally control cellular populations of interest with only magnetically inert materials that do not interact significantly with radiofrequency pulses makes optogenetics incredibly attractive for combination with MRI experiments. However, visible light has relatively poor penetration through tissue. Accordingly, to generate the 1–10 mW/mm2 of light power flux necessary to activate optogenetic molecular devices, light needs to be locally delivered to the desired area. Most optogenetics experiments solve this problem by implanting a guide cannula targeted to the region of interest such that a fiber optic cable connected to a light source, such as a laser or high power LED, may be transmitted through the channel to an appropriate depth reproducibly. Considerations in this regard are whether the fiber optic system of choice has a numerical aperture and optical diameter appropriate for the geometry of the tissue region of interest. Detailed protocols for such surgery and optical stimulation are available (43). Combining optogenetics manipulations simultaneously with fMRI, to investigate the function and connectivity of brain circuits, has been performed in rats that have a specific population of (excitatory) neurons which were engineered via virus transfection to express specific opsins (ChR2 and eNpHR3), and in transgenic mice expressing ChR2. Figure 1 shows a schematic illustration of the experimental setup that we have built for the animal dedicated MRI system that includes a dedicated holder for the optic fiber and the surface coil. In order to illuminate the primary somatosensory cortex (S1), we have thinned the skull above S1 and mounted a 400 micron-diameter optic fiber coupled to a laser source over the in the center of the craniotomy. The craniotomy window was then filled with 2% agarose gel in order to minimize susceptibility artifacts. For optogenetics manipulations of deep brain areas such as the thalamus, the optic fiber was inserted through an implanted guiding cannula (44).

Figure 1. A schematic illustration and photo of the experimental setup for the animal dedicated MRI system that includes a dedicated holder for the optic fiber and the surface coil.

Figure 1

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Putting all these considerations together towards a scientific goal may be technically challenging in initial stages, to be sure. The power of controlling a variety of cellular processes in a genetically-specific cellular population with high temporal resolution, and to do so with advanced MRI techniques as a readout of the global effects of such cellular stimulation, will allow for a revolution in our understanding of the in vivo effects of these cellular processes, as we discuss further in the next section.

Optogenetics and functional MRI

The brain has the tremendous capability to adapt itself in response to internal and external events. The ability of neurons to change their internal properties, and of neuronal networks to reshape their connections, is referred to as plasticity. The outcome of these plasticity changes can affect the time it takes the brain to process a specific stimulation, and generate a suitable response. Therefore, appropriate rewiring of neuronal connections during development and in adulthood is crucial to ensure proper and adequate propagation and processing of stimuli. One of the fundamental goals in neuroscience is to determine the genetic, epigentic, cellular, systemic and environmental basis of plasticity changes. BOLD fMRI techniques enable for the detection of hemodynamic changes due to changes in neural activity throughout the brain. In that respect, human fMRI has had a major impact in cognitive neuroscience and neurosurgery planning where an emphasis is on the role of plasticity in recovery and maintenance of brain functions in a wide range of diseases. Indeed, a revolution has occurred indicating that extensive and widespread plasticity takes place in the adult brain.

The ability to manipulate normal neuronal functions in rodents using genetic, molecular biology and neurosurgical tools and to monitor the consequent changes in neuronal behavior make rodent models the primary preference when investigating the underlying mechanisms of neuronal plasticity. Over the past decade, new developments in MRI of rodents have enabled spatial resolution of approximately 100 microns and temporal resolution for functional changes on the order of 500 ms, making MRI an emerging tool for studying plasticity in animal models. Changes in the spatial localization and the magnitude of BOLD fMRI responses were observed in rodent models following lesions in the central nervous system (45–47) and injuries of the peripheral nervous system (48–51). Similar to human fMRI studies, it is apparent that reorganization of neuronal pathways following injury in the rodent brain is reflected by the fMRI responses.

Nevertheless, the BOLD fMRI signal is an indirect measurement of neuronal activity. The exact relationship between the neuronal responses and hemodynamic responses remains unclear and under debate (52–58). Indeed in recent years there have been great efforts to resolve this controversial topic. The vast majority of the microscopic research is focused on exploring the molecular factors implicated in the underlying neurovascular coupling, such as vasoactive ions, vasoactive factors related to energy metabolism, vasoactive factors/neurotransmitters released by neuronal activation, and the role astrocytes play in neurovascular coupling (59–62). The majority of the macroscopic research is focused on investigating the temporal correlation between the magnitude of the neuronal responses and the hemodynamic responses. Studies have demonstrated that the BOLD fMRI responses are tightly correlated to increases in local field potential (LFP) and spiking activity (63–67); on the other hand, dissociations between LFP and spiking activity to hemodynamic responses have been reported (68–70). Understanding this relationship is crucial to define boundaries of cortical representations of any stimulus or response. Such knowledge, for example, would be useful in determining the degree of plasticity associated with development and injury.

Currently, electrical stimulation via electrodes is used to map and modulate the stimulus response in the brain in combination with fMRI measurements in non-primates and rodents (71–73). However, stimulating electrodes to induce specific neuronal responses or measure connectivity of brain regions can confound functional mapping since the electrical activation recruits a heterogeneous population of excitatory, inhibitory and modulatory cells. In addition, using stimulation electrodes in combination of MRI often generate susceptibility artifacts in the MR image. As discussed previously, optogenetic tools now enable neuronal manipulations that are precise, reversible and cell specific. In combination with MRI, optogenetics emerges as a powerful tool to investigate detailed neuronal mechanisms associated with brain function. For example, optogenetic tools are being utilized to address questions regarding the basis of the underlying neuronal activity in BOLD fMRI signals that were challenging to measure before.

In a recent study, light stimulation of ChR2 expressing excitatory neurons in the primary motor cortex (M1) and thalamus during fMRI acquisition was performed in rats. The optogenetics stimulation have generated BOLD fMRI responses in the light stimulated areas whose time courses matched responses of conventional sensory-evoked stimulation, demonstrating that firing of excitatory neurons are involved in the neurovascular coupling driving the hemodynamic responses (44). As shown in Figure 2, we demonstrated that light stimulation of ChR2 (inducing neuronal firing) and of eNpHR (silencing neuronal firing) expressing excitatory neurons in the primary somatosensory cortex (S1) is capable of modulating the sensory-evoked BOLD fMRI responses in rats (74,75).

Figure 2. Modulations in the BOLD fMRI responses in the rat’s primary somatosensory cortex (S1) induced by optogenetics stimulation.

Figure 2

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a. Examples of BOLD fMRI activation z-maps (p<0.05) induced by sensory (contralateral forepaw) and ChR2 stimulation overlaid on the EPI images. The optic fiber was placed directly above the right S1. The time courses of BOLD fMRI responses across the different cortical laminae are shown. b. Light induced activation of eNpHR resulted in decreases in both the extent and the amplitude of BOLD fMRI responses during forepaw stimulation mainly in the upper cortical laminae. Z statistic activation maps (p<0.05) are overlaid on RARE anatomical images. Red bars represent forepaw stimulation. Images were acquired using a Bruker 9.4 T animal dedicated scanner.

The combinations of optogenetics with fMRI can also facilitate detailed investigation of brain connectivity and function of neuronal circuits associated with plasticity and brain pathologies. Light stimulation of ChR2 expressing excitatory neurons in M1 resulted in BOLD fMRI responses in downstream regions (thalamus) demonstrating their strong connectivity (44). In addition, anesthesia has been shown to dramatically decrease the optogenetics-induced BOLD fMRI connectivity between cortical and sub-cortical areas in mice (76).

The BOLD fMRI temporal and laminar characteristics of light stimulation of ChR2 expressing excitatory neurons in lamina V of S1 of mice were compared to conventional vibrissa (sensory) stimulation. Both stimulation modalities gave rise to identical BOLD fMRI and electrophysiological responses (77). To date, the groups that combined optogenetics with fMRI used different animal models, the light sensitive channels were expressed in different neuronal populations and the optogenetics stimulations took place in different sites. Thus, careful interpretation of the findings, in terms of the function of the neuronal circuitry and the neuronal basis of the BOLD fMRI signals, should be employed.

In our studies, we also used BOLD fMRI as a mean to monitor optogenetics-induced changes in post-injury plasticity in a rat model for peripheral nerve injury. Through optogenetics manipulation of cortical neurons in the hemisphere ipsilateral to the injured forepaw, we guided the cortical reorganization (35). As shown in Figure 3, we successfully decreased the inhibition in S1 ipsilateral to the injured forepaw, which in turn could facilitate recovery and rehabilitation following peripheral nerve injury.

Figure 3. BOLD fMRI shows increased responses within the primary somatosensory cortex (S1) contralateral to the injured forepaw during eNpHR stimulation.

Figure 3

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a. In denervated rats, stimulation of eNpHR of the healthy (right) S1 induced increases in BOLD fMRI responses in the deprived S1, ipsilateral to intact forepaw stimulation. Z-maps (p<0.05) are overlaid on RARE anatomical images. b. Group average of the BOLD fMRI response spatial extent in S1 contralateral (C) and ipsilateral (I) to forepaw stimulation in control (n=5) and denervated (n=5) rats with or without eNpHR stimulation (*, p<0.05). Images were acquired using a Bruker 9.4 T animal dedicated scanner.

This work strengthens the notion that the combination of optogenetics and fMRI can provide an appropriate platform to resolve pre-synaptic vs. post-synaptic changes of neuronal activity and changes in the activity of large neuronal networks in the context of plasticity associated with development, learning and injury.

In summary, the application of novel genetic tools that can manipulate the MR contrast can open a new avenue for understanding complex biological and physiological systems.

Acknowledgments

GP is thankful to Bruker for their collaborative support on this work, and David Glover for assisting with manuscript preparation. This work was supported by NIH/NINDS 5R01NS072171.

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