Blood oxygenation level-dependent functional magnetic resonance imaging (BOLD fMRI)1
is a widely used technology for non-invasive whole brain imaging. BOLD signals reflect complex and incompletely understood changes in cerebral blood flow (CBF), cerebral blood volume (CBV), and cerebral metabolic rate of oxygen consumption (CMRO2
) following neuronal activity2,15
. Candidate circuit elements for triggering various kinds of BOLD signals include excitatory neurons, mixed neuronal populations, astroglia, and axonal tracts or fibers of passage16,17
. Importantly, it is not clear which kinds of activity are capable of triggering BOLD responses, placing limitations on interpretation for both clinical and scientific applications. For example, it is sometimes assumed that positive BOLD signals can be triggered by increased activity of local excitatory neurons, but this remains to be shown empirically, a challenge which seriously confounds fMRI interpretation18,19
. Moreover, the use of MRI-compatible electrodes for local stimulation, while of pioneering significance, will nevertheless drive all local excitatory, inhibitory, and modulatory cell types, as well as antidromically drive non-local cells that happen to have axons within the stimulated region, thereby confounding functional circuit mapping using BOLD. We sought to address these challenges by integrating high-field fMRI output with optogenetic stimulation4–13
, in which single-component microbial light-activated transmembrane conductance regulators are introduced into specifically targeted cell types and circuit elements7,8
using cell type-specific promoters to allow millisecond-scale targeted activity modulation in vivo9–11
In adult rats, primary motor cortex (M1) was injected with the adeno-associated viral vector AAV5-CaMKIIα::ChR2(H134R)-EYFP to drive expression of a channelrhodopsin (ChR2) specifically in CaMKIIα-expressing principal cortical neurons, but not in GABAergic or glial cells7,9–12
; here the cortical virus injection site was also employed as the optical stimulation site for BOLD and electrophysiological functional studies (). To minimize susceptibility artifact during MRI scanning, the implanted cannula, optical fiber and accessories were custom-fabricated from MR-compatible materials. Confocal imaging () and optrode recording (simultaneous optical stimulation and electrical recording) under 1.3–1.5% isoflurane anaesthesia20
() were conducted to validate the expression and functionality, respectively, of ChR2-EYFP under these conditions; in line with previous optogenetic studies11
, 473 nm light pulses at 20 Hz (15 ms pulse width) delivered through the optical fiber were found to reliably drive local neuronal firing () in vivo
ofMRI: optogenetic excitation of CaMKIIα neocortical cells drives local positive BOLD
To assess fMRI signals, we acquired 0.5 mm coronal slices centered on M1, >10 days after virus injection (). Intubated animals were placed on a custom-designed MRI-compatible cradle with a stereotaxic frame and ventilated with 1.3–1.5% isoflurane. A 3.5 cm-diameter custom-designed transmit/receive single-loop surface coil was apposed to the cranium and a long 300 μm diameter optical fiber inserted through the implanted cannula; in this configuration, the cradle with the animal was placed into the iso-center of the magnet while the laser diode itself was maintained outside the 5 Gauss perimeter. In order to minimize systemic physiological confounds, the ventilation volume, frequency, endtidal CO2
, and rectal temperature levels were carefully maintained at narrow levels known to produce most robust and reproducible BOLD signals in anesthetized animals (3.0–3.5 cc/stroke, 50–60 stroke/min, 3.5%, 34–38°C)20
. fMRI scans were performed at 7.0 Tesla (T) field strength using conventional GRE-BOLD fMRI and passband b-SSFP fMRI21,22
). Both pulse sequences were designed to have 3.5 × 3.5 cm2
in-plane FOV, 0.5 × 0.5 × 0.5 mm3
spatial resolution and 3 s temporal resolution. GRE-BOLD fMRI was designed to be a 2D, multi-slice, gradient-echo (GRE) sequence with 4-interleave spiral readout23
; 750 ms TR
and 12 ms TE
resulting in 23 slices covering 1.15 cm slice direction volume. This specific design allowed large-volume mapping of the brain during optogenetic control with high temporal resolution.
473 nm light pulses at 20 Hz (15 ms pulse width) were delivered to targeted CaMKIIα-expressing principal neurons, and in response, robust optically-evoked BOLD signals were observed in cortical gray matter at the virus injection and optical stimulation site, while in control animals (injected with saline instead of opsin-AAV) no detectable BOLD signal could be elicited (). Stimulus-synchronized BOLD hemodynamic responses from activated M1 voxels are displayed in , and mean optogenetic fMRI hemodynamic response functions (ofMRI-HRF) in . Evoked BOLD was dominated by positive signals while driving these excitatory CaMKIIα-positive cells; in contrast, optically driving inhibitory parvalbumin-positive cells10
, which may have unique connectivity with local neuronal circuitry or vasculature, additionally gave rise to a zone of negative BOLD, consistent with the GABAergic phenotype, surrounding the local positive BOLD signal (Supplementary Fig. 4
). Strikingly, the BOLD dynamics observed by optically driving the defined CaMKIIα principal cell population embedded within the mixed M1 cell population precisely matched dynamics of conventional stimulus-evoked BOLD-fMRI24,25
. In particular, the ofMRI-HRF signal onset occurred after 3 seconds but within 6 seconds of stimulus onset; likewise offset was reflected by a drop in BOLD signal contrast beginning within 6 seconds and returning to baseline in ~20 seconds after optical stimulation (; upper panel: n=3, lower panel: n=8). Finally, the pronounced post-stimulus undershoot observed during systemic somatosensory stimulation in humans26
was preserved in ofMRI-HRFs as well (). All of these dynamic properties derived from driving a defined, specific (Methods; Supplementary Figure 1a
) cell population correspond closely to prior measurements on conventional sensory-evoked BOLD.
In order to study macrocircuit properties of the brain using optogenetic fMRI, it will be important to assess feasibility of monitoring long-range activity in synaptically connected brain areas. MRI-compatible electrodes for local stimulation represent a major advance but in addition to driving all local excitatory, inhibitory, and modulatory cell types, will also antidromically drive non-local cells that happen to have axons within the stimulated region, posing a challenge for functional mapping using BOLD. We therefore employed high-resolution fMRI slices capturing thalamic nuclei (coronal slices shown in ) to monitor downstream responses during optical stimulation of M1 cortical neurons. illustrates the observed specific expression of ChR2(H134R)-EYFP in cortico-thalamic axonal projection fibers while thalamic cell bodies revealed no ChR2 expression, as expected from the cortical injection protocol (Supplementary Fig. 5
). Local optical stimulation was then delivered to cortex during fMRI, to determine if unidirectionally triggered BOLD responses could be observed and measured (this method eliminates the antidromic drive confound from which electrical stimulation suffers, thereby allowing true global causal connectivity mapping). summarize the thalamic ofMRI-HRFs; robust thalamic BOLD signals in response to M1 stimulation were observed, but with properties quite distinct from the intracortical CaMKIIα + response described above. A markedly reduced initial rise and slope for onset kinetics of positive-BOLD downstream thalamic recruitment was observed (, black traces; local cortical BOLD signals shown for comparison, gray traces; cortical BOLD activation is shown in ).
Nonlocal mapping of causal role of cells defined by location and genetic identity
Given the unusual kinetics, we sought to determine if this delayed thalamic BOLD response would be discrepant with local thalamic electrical activity, assessed with simultaneous optrode stimulating/recording in motor cortex and electrode recoding in thalamus (). However, a strikingly similar pattern was observed with direct recording in thalamus, including a commensurate delay in spike-rate increase for thalamic neurons compared to cortical neurons during cortical optogenetic drive (), further supporting the tight correspondence between positive BOLD and local neuronal excitation2,27,28
. Additional characterization revealed that after this ~5 second delay presumably related to network properties, successfully evoked spikes recorded in thalamus reliably followed cortical spikes by several milliseconds, as expected (). Summary data on mean spike rates is presented in , and on spike rate dynamics in ; further details on the passband bSSFP-fMRI21,22
method we developed for small animal imaging with more robust whole-brain mapping capability than traditional BOLD are presented in the Supplementary Material (Supplementary Figure 3)
Since true functional outputs of genetically defined neurons in a brain region can be globally mapped with ofMRI (), it is conceivable that additional levels of specificity could also be achieved. For example, M1 excitatory pyramidal neurons form a genetically- and anatomically-defined class of cell, but within this class are cells that each project to different areas of the brain or spinal cord and therefore have fundamentally distinct roles. Genetic tools may not advance far enough to separate all of these different cell classes, pointing to the need for other promoter-independent targeting methods13
. But ofMRI raises the current possibility of globally mapping the casual roles of these cells, accessing them by means of connection topology—i.e. by the conformation of their functional projection patterns in the brain. We therefore sought to test this possibility by selectively driving the M1 CaMKIIα-expressing cells that project to thalamus.
An optical fiber was stereotactically placed in thalamus of animals that had received M1 cortical viral injections (); posthoc validation of ChR2 expression () confirmed ChR2–YFP in cortical neurons and in cortico-thalamic projection fibers. ChR2 readily triggers spikes in illuminated photosensitive axons, that both drive local synaptic output and back-propagate throughout the axon, to the soma of the stimulated cell; note that unlike the case with electrical stimulation, specificity is maintained for driving the targeted (photosensitive) axons, and therefore this configuration in principle allows ofMRI mapping during selective control of the M1 cortical cells that project to thalamus. Indeed robust BOLD signals were observed both locally in thalamus (: coronal slices 7–12) and also in M1 (: coronal slices 1–6), consistent with the anticipated recruitment of the topologically targeted cells both locally and distally. These data demonstrate that ChR2-expressing axonal fiber stimulation alone is sufficient to elicit BOLD responses in remote areas, and illustrate the feasibility for in vivo mapping of the global impact of cells defined not only by anatomical location and genetic identity, but also by connection topology.
Control of cells defined by location, genetic identity, and wiring during ofMRI
We further explored the global mapping capabilities of ofMRI. It has been suggested that thalamic projections to motor cortex may be more likely than those to sensory cortex, to involve both ipsilateral and contralateral pathways, since in many cases motor control and planning must involve bilateral coordination29
. This principle is challenging to assess at the functional level, since electrode-based stimulation will drive antidromic as well as orthodromic projections, and hence may mistakenly report robust cortico-thalamic rather than thalamocortical projections. We therefore sought to globally map functional connectivity arising from initial drive of anterior or posterior thalamic nucleus projections, employing ofMRI. After injecting CaMKIIα::ChR2 into thalamus (), we found that optical stimulation of posterior thalamic nuclei resulted in a strong BOLD response, both at the site of stimulation as expected and in the posterior ipsilateral somatosensory cortex (S2) (). Optically stimulating excitatory cell bodies and fibers in the more anterior thalamic nuclei resulted in BOLD response at the site of stimulation and also significant ipsilateral and contralateral cortical BOLD responses (), consistent with the proposed bilaterality of anterior thalamocortical nuclei involvement in motor control and coordination30
Recruitment of bilateral cortices by anterior thalamus
Together, these results illustrate the power of optogenetic fMRI in shedding light on the controversial identification of positive BOLD signals with increased local neuronal excitation, providing empirical underpinning for fMRI BOLD. We also find that the properties of integrated optogenetics and BOLD-fMRI (ofMRI) allow for global mapping of the causal connectivity of defined neurons in specific brain regions, fundamentally extending the capabilities of pharmacological or electrode-based methods (of course, contributions from additional cells and processes downstream of the defined optically-triggered population are expected and indeed represent an important aspect of this approach; it is, however, important to note that absence of a BOLD signal does not prove the absence of connectivity). Finally, we demonstrate that ofMRI allows causal connectivity mapping of cells defined not only genetically but also by circuit topology, or the conformation of their connections in vivo. Together, the ofMRI methods and findings reported here provide tools and approaches for further probing and defining the causal generation of BOLD signals; these results may accelerate the search for global circuit-disease endophenotypes, as well as the dynamical mapping and reverse engineering of intact neural circuitry.