Odor-evoked layer-specific fMRI activities in the awake mouse olfactory bulb.

Awake rodent fMRI is increasingly common over the use of anesthesia since it permits behavioral paradigms and does not confound normal brain function or neurovascular coupling. It is well established that adequate acclimation to the loud fMRI environment and head fixation reduces stress in the rodents and allows for whole brain imaging with little contamination from motion. However, it is unknown whether high-resolution fMRI with increased susceptibility to motion and lower sensitivity can measure small, but spatially discrete, activations in awake mice. To examine this, we used contrast-enhanced cerebral blood volume-weighted (CBVw) fMRI in the mouse olfactory bulb for its enhanced sensitivity and neural specificity. We determined that activation patterns in the glomerular layer to four different odors were spatially distinct and were consistent with previously established histological patterns. In addition, odor-evoked laminar activations were greatest in superficial layers that decreased with laminar depth, similar to previous observations. Interestingly, the fMRI response strengths in the granule cell layer were greater in awake mice than our previous anesthetized rat studies, suggesting that feedback neural activities were intact with wakefulness. We finally determined that fMRI signal changes to repeated odor exposure (i.e., olfactory adaptation) attenuated relatively more in the feedback granule cell layer compared to the input glomerular layer, which is consistent with prior observations. We, therefore, conclude that high-resolution CBVw fMRI can measure odor-specific activation patterns and distinguish changes in laminar activity of head and body restrained awake mice.

Physical therapy exerts sub-additive and suppressive effects on intracerebral neural stem cell implantation in a rat model of stroke.

Intracerebral cell therapy (CT) is emerging as a new therapeutic paradigm for stroke. However, the impact of physical therapy (PT) on implanted cells and their ability to promote recovery remains poorly understood. To address this translational issue, a clinical-grade neural stem cell (NSC) line was implanted into peri-infarct tissue using MRI-defined injection sites, two weeks after stroke. PT in the form of aerobic exercise (AE) was administered 5 × per week post-implantation using a paradigm commonly applied in patients with stroke. A combined AE and CT exerted sub-additive therapeutic effects on sensory neglect, whereas AE suppressed CT effects on motor integration and grip strength. Behavioral testing emerged as a potentially major component for task integration. It is expected that this study will guide and inform the incorporation of PT in the design of clinical trials evaluating intraparenchymal NSCs implantation for stroke.

Postsynaptic activity of inhibitory neurons evokes hemodynamic fMRI responses.

Functional MRI responses are localized to the synaptic sites of evoked inhibitory neurons, but it is unknown whether, or by what mechanisms, these neurons initiate functional hyperemia. Here, the neuronal origins of these hemodynamic responses were investigated by fMRI or local field potential and blood flow measurements during topical application of pharmacological agents when GABAergic granule cells in the rat olfactory bulb were synaptically targeted. First, to examine if postsynaptic activation of these inhibitory neurons was required for neurovascular coupling, we applied an NMDA receptor antagonist during cerebral blood volume-weighted fMRI acquisition and found that responses below the drug application site (up to ~1.5 mm) significantly decreased within ~30 min. Similarly, large decreases in granule cell postsynaptic activities and blood flow responses were observed when AMPA or NMDA receptor antagonists were applied. Second, inhibition of nitric oxide synthase preferentially decreased the initial, fast component of the blood flow response, while inhibitors of astrocyte-specific glutamate transporters and vasoactive intestinal peptide receptors did not decrease blood flow responses. Third, inhibition of GABA release with a presynaptic GABAB receptor agonist caused less reduction of neuronal and blood flow responses compared to the postsynaptic glutamate receptor antagonists. In conclusion, local hyperemia by synaptically-evoked inhibitory neurons was primarily driven by their postsynaptic activities, possibly through NMDA receptor-dependent calcium signaling that was not wholly dependent on nitric oxide.

Dominance of layer-specific microvessel dilation in contrast-enhanced high-resolution fMRI: Comparison between hemodynamic spread and vascular architecture with CLARITY.

Contrast-enhanced cerebral blood volume-weighted (CBVw) fMRI response peaks are specific to the layer of evoked synaptic activity (Poplawsky et al., 2015), but the spatial resolution limit of CBVw fMRI is unknown. In this study, we measured the laminar spread of the CBVw fMRI evoked response in the external plexiform layer (EPL, 265 ± 65 µm anatomical thickness, mean ± SD, n = 30 locations from 5 rats) of the rat olfactory bulb during electrical stimulation of the lateral olfactory tract and examined its potential vascular source. First, we obtained the evoked CBVw fMRI responses with a 55 × 55 µm2 in-plane resolution and a 500-µm thickness at 9.4 T, and found that the fMRI signal peaked predominantly in the inner half of EPL (136 ± 54 µm anatomical thickness). The mean full-width at half-maximum of these fMRI peaks was 347 ± 102 µm and the functional spread was approximately 100 or 200 µm when the effects of the laminar thicknesses of EPL or inner EPL were removed, respectively. Second, we visualized the vascular architecture of EPL from a different rat using a Clear Lipid-exchanged Anatomically Rigid Imaging/immunostaining-compatible Tissue hYdrogel (CLARITY)-based tissue preparation method and confocal microscopy. Microvascular segments with an outer diameter of <11 µm accounted for 64.3% of the total vascular volume within EPL and had a mean segment length of 55 ± 40 µm (n = 472). Additionally, vessels that crossed the EPL border had a mean segment length outside of EPL equal to 73 ± 61 µm (n = 28), which is comparable to half of the functional spread (50-100 µm). Therefore, we conclude that dilation of these microvessels, including capillaries, likely dominate the CBVw fMRI response and that the biological limit of the fMRI spatial resolution is approximately the average length of 1-2 microvessel segments, which may be sufficient for examining sublaminar circuits.

Foundations of layer-specific fMRI and investigations of neurophysiological activity in the laminarized neocortex and olfactory bulb of animal models.

Laminar organization of neuronal circuits is a recurring feature of how the brain processes information. For instance, different layers compartmentalize different cell types, synaptic activities, and have unique intrinsic and extrinsic connections that serve as units for specialized signal processing. Functional MRI is an invaluable tool to investigate laminar processing in the in vivo human brain, but it measures neuronal activity indirectly by way of the hemodynamic response. Therefore, the accuracy of high-resolution laminar fMRI depends on how precisely it can measure localized microvascular changes nearest to the site of evoked activity. To determine the specificity of fMRI responses to the true neurophysiological responses across layers, the flexibility to invasive procedures in animal models has been necessary. In this review, we will examine different fMRI contrasts and their appropriate uses for layer-specific fMRI, and how localized laminar processing was examined in the neocortex and olfactory bulb. Through collective efforts, it was determined that microvessels, including capillaries, are regulated within single layers and that several endogenous and contrast-enhanced fMRI contrast mechanisms can separate these neural-specific vascular changes from the nonspecific, especially cerebral blood volume-weighted fMRI with intravenous contrast agent injection. We will also propose some open questions that are relevant for the successful implementation of layer-specific fMRI and its potential future directions to study laminar processing when combined with optogenetics.

Layer-Specific fMRI Responses to Excitatory and Inhibitory Neuronal Activities in the Olfactory Bulb.

High-resolution functional magnetic resonance imaging (fMRI) detects localized neuronal activity via the hemodynamic response, but it is unclear whether it accurately identifies neuronal activity specific to individual layers. To address this issue, we preferentially evoked neuronal activity in superficial, middle, and deep layers of the rat olfactory bulb: the glomerular layer by odor (5% amyl acetate), the external plexiform layer by electrical stimulation of the lateral olfactory tract (LOT), and the granule cell layer by electrical stimulation of the anterior commissure (AC), respectively. Electrophysiology, laser-Doppler flowmetry of cerebral blood flow (CBF), and blood oxygenation level-dependent (BOLD) and cerebral blood volume-weighted (CBV) fMRI at 9.4 T were performed independently. We found that excitation of inhibitory granule cells by stimulating LOT and AC decreased the spontaneous multi-unit activities of excitatory mitral cells and subsequently increased CBF, CBV, and BOLD signals. Odor stimulation also increased the hemodynamic responses. Furthermore, the greatest CBV fMRI responses were discretely separated into the same layers as the evoked neuronal activities for all three stimuli, whereas BOLD was poorly localized with some exception to the poststimulus undershoot. In addition, the temporal dynamics of the fMRI responses varied depending on the stimulation pathway, even within the same layer. These results indicate that the vasculature is regulated within individual layers and CBV fMRI has a higher fidelity to the evoked neuronal activity compared with BOLD. Our findings are significant for understanding the neuronal origin and spatial specificity of hemodynamic responses, especially for the interpretation of laminar-resolution fMRI. SIGNIFICANCE STATEMENT: Functional magnetic resonance imaging (fMRI) is a noninvasive, in vivo technique widely used to map function of the entire brain, including deep structures, in animals and humans. However, it measures neuronal activity indirectly by way of the vascular response. It is currently unclear how finely the hemodynamic response is regulated within single cortical layers and whether increased inhibitory neuronal activities affect fMRI signal changes. Both laminar specificity and the neural origins of fMRI are important to interpret functional maps properly, which we investigated by activating discrete rat olfactory bulb circuits.

Layer-dependent BOLD and CBV-weighted fMRI responses in the rat olfactory bulb.

The olfactory bulb is a laminarized brain structure involved in odor sensation that has important implications to basic neuroscience research, like mechanisms for neurovascular coupling and early disease diagnosis. To investigate laminar-dependent responses to odor exposure, blood oxygenation level-dependent (BOLD) and cerebral blood volume weighted (CBVw) fMRI with iron oxide nanoparticle contrast agent were obtained with 110×110×500µm(3) resolution in urethane-anesthetized rats at 9.4T. The baseline total CBV is the largest at the olfactory bulb surface and midline, and decreases in the deeper layers, while a band of increased microvasculature density is observed at the glomerular, external plexiform and mitral cell layers. With odor exposure, CBVw fMRI is more sensitive and reproducible than BOLD. BOLD fMRI had the greatest activation on the bulb surface, midline, olfactory nerve and glomerular layers, while CBVw activation peaked in glomerular and external plexiform layers, but was still significant in mitral cell layer. Negative BOLD responses were observed in the bulb midline and near large blood vessels. CBVw laminar profiles are similar to the layer-dependent metabolic changes to the same odor exposure reported by previous glucose metabolism studies. Unique activation patterns for two different odor conditions were also differentiated with CBVw fMRI. Our study suggests that CBVw activation better represents the spatial location of metabolic activity in the olfactory bulb than BOLD.