Layer-dependent fMRI allows measurements of information flow in cortical circuits, as afferent and efferent connections terminate in different cortical layers. However, it is unknown to what level human fMRI is specific and sensitive enough to reveal directional functional activity across layers. To answer this question, we developed acquisition and analysis methods for blood-oxygen-level-dependent (BOLD) and cerebral-blood-volume (CBV)-based laminar fMRI and used these to discriminate four different tasks in the human motor cortex (M1). In agreement with anatomical data from animal studies, we found evidence for somatosensory and premotor input in superficial layers of M1 and for cortico-spinal motor output in deep layers. Laminar resting-state fMRI showed directional functional connectivity of M1 with somatosensory and premotor areas. Our findings demonstrate that CBV-fMRI can be used to investigate cortical activity in humans with unprecedented detail, allowing investigations of information flow between brain regions and outperforming conventional BOLD results that are often buried under vascular biases.

The persistent coupling of the BOLD signal to the field potential when LFP and MUA have different time evolutions suggests that BOLD is primarily determined by the local processing of inputs in a given cortical area. In the alert animal the largest portion of the BOLD signal's variance is explained by an LFP range (20-60 Hz) that is most likely related to neuromodulation. Finally, the similarity of the results in alert and anesthetized subjects indicates that at least in V1 anesthesia is not a confounding factor. This enables the comparison of human fMRI results with a plethora of electrophysiological results obtained in alert or anesthetized animals.

Recurrence 1Electrical stimulation (ES) is used in animals and humans to study potential causal links between neural activity and specific cognitive functions. Recently, it has found increased application in electrotherapy and neural prostheses as well. However, how ES-elicited signals propagate in brain tissues is still unclear. Here we used combined electrostimulation, neurophysiology, microinjection and fMRI to study the cortical activity patterns elicited during stimulation of cortical afferents in monkeys. We find that stimulation of a site in LGN (lateral geniculate nucleus) increases the fMRI signal in the regions of primary visual cortex (V1) receiving input from that site, but suppresses it in the retinotopically matched regions of extrastriate cortex. In agreement with previous observations, intracranial recordings show that immediately after a stimulation pulse a long-lasting inhibition follows a short excitatory response. Following microinjections of GABA (γ-aminobutyric acid) antagonists in V1, LGN stimulation induces positive fMRI signals in all cortical areas. Taken together, our findings suggest that ES disrupts cortico-cortical signal propagation by silencing the output of any neocortical area whose afferents are electrically stimulated.We recently developed and optimized the esfMRI (combined ES and fMRI) methodology for experiments in anesthetized and behaving monkeys 1, 2 . Our first experiments, including fMRI-based estimations of tissue excitability (rheobase and chronaxie measurements), showed that electrical stimulation of the primary visual cortex V1 mainly excites large pyramidal cells and axons, eliciting positive BOLD responses (PBR) in topographically matched regions of extrastriate areas such as V2, V3, V3A, V4, and MT (V5); all monosynaptic targets of the primary visual cortex. These findings are consistent with the well-established anatomical connections between V1 and the extrastriate cortex of macaque monkeys 3 . One puzzling observation in our initial studies was the clear lack of transsynaptic effects during cortical stimulation. In the present study, we stimulated either the LGN or the pulvinar (Pul) in anesthetized and alert monkeys in order to systematically examine the propagation of ES-induced signals.We demonstrate that electrical stimulation of a thalamic site indeed suppresses the neural activity of its projection regions in visual cortex. The strong reduction in BOLD response is likely due to synaptic inhibition and it be can be reversed by injections of GABA antagonists in V1. In agreement with the fMRI results, intracortical recordings show that an electric pulse evokes an action potential followed by a pronounced and long-lasting inhibition. Such disruptive effects of cortical afferent stimulation on the activity of projection neurons have already been reported. Yet, by using the combined physiology, pharmacology and fMRI approach here we illustrate for the first time the extent and generality of ES-induced activity suppression, and we propose that many behavioral e...

SUMMARY The six cortical layers have distinct anatomical and physiological properties, like different energy use and different feedforward and feedback connectivity. It is not known if and how layer-specific neural processes are reflected in the fMRI signal. To address this question we used high-resolution fMRI to measure BOLD, CBV, and CBF responses to stimuli that elicit positive and negative BOLD signals in macaque primary visual cortex. We found that regions with positive BOLD responses had parallel increases in CBV and CBF, whereas areas with negative BOLD responses showed a decrease in CBF but an increase in CBV. For positive BOLD responses, CBF and CBV increased in the center of the cortex, but for negative BOLD responses, CBF decreased superficially while CBV increased in the center. Our findings suggest different mechanisms for neurovascular coupling for BOLD increases and decreases, as well as laminar differences in neurovascular coupling.

Highlights: 21• A CBV-sensitive fMRI method is developed for high resolution fMRI in humans. 22• Lamina-dependent CBV fMRI responses are shown in humans. 23• VASO cortical profiles are validated with Fe-contrast agent fMRI in animals. 24• Sensitivity to large veins can be minimized using VASO-CBV instead of BOLD fMRI. 25• Ipsilateral fMRI responses to finger-tapping are positive in M1 and negative in S1. 26 27 Abstract 28Cortical layer-dependent high (sub-millimeter) resolution functional magnetic resonance imaging 29 (fMRI) in human or animal brain can be used to address questions regarding the functioning of 30 cortical circuits, such as the effect of different afferent and efferent connectivity on activity in 31 specific cortical layers. The sensitivity of gradient echo (GE) blood oxygenation level dependent 32 (BOLD) responses to large draining veins reduces its local specificity and can render the 33 interpretation of the underlying laminar neural activity impossible. Application of the more spatially 34 specific cerebral blood volume (CBV) based fMRI in humans has been hindered by the low sensitivity 35 of the non-invasive modalities available. Here, a Vascular Space Occupancy (VASO) variant, adapted 36 for use at high field, is further optimized to capture layer-dependent activity changes in human 37 motor cortex at sub-millimeter resolution. Acquired activation maps and cortical profiles show that 38 the VASO signal peaks in grey matter at 0.8 -1.6 mm depth, and deeper compared to the superficial 39 and vein-dominated GE-BOLD responses. Validation of the VASO signal change versus well-40 established iron-oxide contrast agent based fMRI methods in animals showed the same cortical 41 profiles of CBV change, after normalization for lamina-dependent baseline CBV. In order to evaluate 42 its potential of revealing small lamina-dependent signal differences due to modulations of the input-43 output characteristics, layer-dependent VASO responses were investigated in the ipsilateral 44 hemisphere during unilateral finger tapping. Positive activation in ipsilateral primary motor cortex 45 *7. Manuscript Click here to view linked References 2 and negative activation in ipsilateral primary sensory cortex were observed. This feature is only 1 visible in high-resolution fMRI where opposing sides of a sulcus can be investigated independently 2 because of a lack of partial volume effects. Based on the results presented here we conclude that 3 VASO offers good reproducibility, high sensitivity, and lower sensitivity than GE-BOLD to changes in 4 larger vessels, making it a valuable tool for layer-dependent fMRI studies in humans. 5Abbreviations: BOLD = blood oxygenation level dependent; CBV = cerebral blood volume; CNR = 6 contrast-to-noise ratio; CSF = cerebrospinal fluid; ΔCBV = change in CBV; EPI = echo planar imaging; 7 Fe = iron; fMRI = functional magnetic resonance imaging; GE = gradient echo; GM = grey matter; ROI 8 = region of interest; SNR = signal-to-noise ratio; SS-SI-VASO = slice-selective slab-inversion VASO...