Pai-Feng Yang

Injury alters intrinsic functional connectivity within the primate spinal cord

Significance This is the first study, to our knowledge, to demonstrate intrinsic functional connectivity within the spinal cords of monkeys, replicating and extending recent similar discoveries in humans. Our observations of spatially correlated patterns of MRI signals at rest indicate that neurons in different parts of the spinal horns show synchronized fluctuations in activity at rest just as found in the brain. Injury to the cord disrupts the integrity of this interhorn connectivity within and across spinal segments, showing these intrinsic correlations have functional relevance and mirror behavioral deficits in the injured monkeys. Quantification of the intrinsic functional connectivity within the spine after injury therefore has considerable potential as an imaging biomarker of spinal cord functional integrity, something long needed in clinical practice. Recent demonstrations of correlated low-frequency MRI signal variations between subregions of the spinal cord at rest in humans, similar to those found in the brain, suggest that such resting-state functional connectivity constitutes a common feature of the intrinsic organization of the entire central nervous system. We report our detection of functional connectivity within the spinal cords of anesthetized squirrel monkeys at rest and show that the strength of connectivity within these networks is altered by the effects of injuries. By quantifying the low-frequency MRI signal correlations between different horns within spinal cord gray matter, we found distinct functional connectivity relationships between the different sensory and motor horns, a pattern that was similar to activation patterns evoked by nociceptive heat or tactile stimulation of digits. All horns within a single spinal segment were functionally connected, with the strongest connectivity occurring between ipsilateral dorsal and ventral horns. Each horn was strongly connected to the same horn on neighboring segments, but this connectivity reduced drastically along the spinal cord. Unilateral injury to the spinal cord significantly weakened the strength of the intrasegment horn-to-horn connectivity only on the injury side and in slices below the lesion. These findings suggest resting-state functional connectivity may be a useful biomarker of functional integrity in injured and recovering spinal cords.

Differential fMRI Activation Patterns to Noxious Heat and Tactile Stimuli in the Primate Spinal Cord

Mesoscale local functional organizations of the primate spinal cord are largely unknown. Using high-resolution fMRI at 9.4 T, we identified distinct interhorn and intersegment fMRI activation patterns to tactile versus nociceptive heat stimulation of digits in lightly anesthetized monkeys. Within a spinal segment, 8 Hz vibrotactile stimuli elicited predominantly fMRI activations in the middle part of ipsilateral dorsal horn (iDH), along with significantly weaker activations in ipsilateral (iVH) and contralateral (cVH) ventral horns. In contrast, nociceptive heat stimuli evoked widespread strong activations in the superficial part of iDH, as well as in iVH and contralateral dorsal (cDH) horns. As controls, only weak signal fluctuations were detected in the white matter. The iDH responded most strongly to both tactile and heat stimuli, whereas the cVH and cDH responded selectively to tactile versus nociceptive heat, respectively. Across spinal segments, iDH activations were detected in three consecutive segments in both tactile and heat conditions. Heat responses, however, were more extensive along the cord, with strong activations in iVH and cDH in two consecutive segments. Subsequent subunit B of cholera toxin tracer histology confirmed that the spinal segments showing fMRI activations indeed received afferent inputs from the stimulated digits. Comparisons of the fMRI signal time courses in early somatosensory area 3b and iDH revealed very similar hemodynamic stimulus–response functions. In summary, we identified with fMRI distinct segmental networks for the processing of tactile and nociceptive heat stimuli in the cervical spinal cord of nonhuman primates. SIGNIFICANCE STATEMENT This is the first fMRI demonstration of distinct intrasegmental and intersegmental nociceptive heat and touch processing circuits in the spinal cord of nonhuman primates. This study provides novel insights into the local functional organizations of the primate spinal cord for pain and touch, information that will be valuable for designing and optimizing therapeutic interventions for chronic pain management.

Parallel functional reorganizations of somatosensory areas 3b and 1, and S2 following spinal cord injury in squirrel monkeys.

Multiple somatosensory cortices of adult primates reorganize following spinal cord injury, but little is known about the temporal dynamics and inter-areal differences of the reorganization. Using longitudinal high-resolution fMRI in combination with microelectrode recordings and tracer histology, we previously illustrated a two-phase dynamic spatial reorganization of digit representations in area 3b within weeks after a unilateral lesion of the dorsal column in squirrel monkeys (Chen et al., 2012). Here we report that higher-order area 1 and secondary somatosensory cortex (S2) underwent similar spatial reorganizations, which were characterized by shifted and expanded digit activations at week 4 after lesion, which then shifted back and contracted by week 8. In addition, the responsiveness of areas 3b and 1, and S2, as measured by the magnitude of the BOLD signal change to tactile stimuli, was reduced markedly at 4 weeks and then recovered to ∼50% of the prelesion level at 8 weeks, a time when behavioral recovery was complete, as assessed by successful food retrieval rates. Across animals, the extents of spatial reorganizations and changes in cortical responsiveness and activation sizes in all three areas were correlated with the degree of afferent disruption. In summary, our data show that more severe afferent disruption was associated with greater cortical plasticity and behavioral impairment. Reorganization that occurred in area 3b, area 1, and S2 were similar across most measures.

Biophysical and neural basis of resting state functional connectivity: Evidence from non-human primates

Functional MRI (fMRI) has evolved from simple observations of regional changes in MRI signals caused by cortical activity induced by a task or stimulus, to task-free acquisitions of images in a resting state. Such resting state signals contain low frequency fluctuations which may be correlated between voxels, and strongly correlated regions are deemed to reflect functional connectivity within synchronized circuits. Resting state functional connectivity (rsFC) measures have been widely adopted by the neuroscience community, and are being used and interpreted as indicators of intrinsic neural circuits and their functional states in a broad range of applications, both basic and clinical. However, there has been relatively little work reported that validates whether inter-regional correlations in resting state fluctuations of fMRI (rsfMRI) signals actually measure functional connectivity between brain regions, or to establish how MRI data correlate with other metrics of functional connectivity. In this mini-review, we summarize recent studies of rsFC within mesoscopic scale cortical networks (100μm-10mm) within a well defined functional region of primary somatosensory cortex (S1), as well as spinal cord and brain white matter in non-human primates, in which we have measured spatial patterns of resting state correlations and validated their interpretation with electrophysiological signals and anatomic connections. Moreover, we emphasize that low frequency correlations are a general feature of neural systems, as evidenced by their presence in the spinal cord as well as white matter. These studies demonstrate the valuable role of high field MRI and invasive measurements in an animal model to inform the interpretation of human imaging studies.

Correlated inter-regional variations in low frequency local field potentials and resting state BOLD signals within S1 cortex of monkeys.

The hypothesis that specific frequency components of the spontaneous local field potentials (LFPs) underlie low frequency fluctuations of resting state fMRI (rsfMRI) signals was tested. The previous analyses of rsfMRI signals revealed differential inter‐regional correlations among areas 3a, 3b, and 1 of primary somatosensory cortex (S1) in anesthetized monkeys (Wang et al. [2013]: Neuron 78:1116–1126). Here LFP band(s) which correlated between S1 regions, and how these inter‐regional correlation differences covaried with rsfMRI signals were examined. LFP signals were filtered into seven bands (delta, theta, alpha, beta, gamma low, gamma high, and gamma very high), and then a Hilbert transformation was applied to obtain measures of instantaneous amplitudes and temporal lags between regions of interest (ROI) digit–digit pairs (areas 3b–area 1, area 3a–area 1, area 3a–area 3b) and digit–face pairs (area 3b–face, area 1–face, and area 3a–face). It was found that variations in the inter‐regional correlation strengths between digit–digit and digit–face pairs in the delta (1–4 Hz), alpha (9–14 Hz), beta (15–30 Hz), and gamma (31–50 Hz) bands parallel those of rsfMRI signals to varying degrees. Temporal lags between digit–digit area pairs varied across LFP bands, with area 3a mostly leading areas 1/2 and 3b. In summary, the data demonstrates that the low and middle frequency range (1–50 Hz) of spontaneous LFP signals similarly covary with the low frequency fluctuations of rsfMRI signals within local circuits of S1, supporting a neuronal electrophysiological basis of rsfMRI signals. Inter‐areal LFP temporal lag differences provided novel insights into the directionality of information flow among S1 areas at rest. Hum Brain Mapp 37:2755–2766, 2016.

Effects of isoflurane anesthesia on resting-state fMRI signals and functional connectivity within primary somatosensory cortex of monkeys.

Correlated low‐frequency fluctuations of resting‐state functional magnetic resonance imaging (rsfMRI) signals have been widely used for inferring intrinsic brain functional connectivity (FC). In animal studies, accurate estimate of anesthetic effects on rsfMRI signals is demanded for reliable interpretations of FC changes. We have previously shown that inter‐regional FC can reliably delineate local millimeter‐scale circuits within digit representations of primary somatosensory cortex (S1) subregions (areas 3a, 3b, and 1) in monkeys under isoflurane anesthesia. The goals of this study are to determine (1) the general effects of isoflurane on rsfMRI signals in the S1 circuit and (2) whether the effects are functional‐ and regional‐ dependent, by quantifying the relationships between isoflurane levels, power and inter‐regional correlation coefficients in digit and face regions of distinct S1 subregions.

High spatial correspondence at a columnar level between activation and resting state fMRI signals and local field potentials

Significance We found that blood oxygenation level-dependent (BOLD) signal changes within single-digit representation columns in the primary somatosensory cortices of areas 3b and 1 aligned spatially very closely with local field potential (LFP) signals in response to tactile stimulation. Moreover, resting state BOLD fMRI and LFP signals also exhibited very similar intervoxel spatial correlation profiles. These findings indicate that at a columnar level, BOLD signals faithfully reflect underlying neuronal activity both during information processing and at rest. Importantly, the spread of BOLD activity and correlations at high field are no greater than the extent of LFP signals. These results demonstrate that high-field fMRI has the ability to delineate brain activity at the columnar level, and BOLD signals faithfully reflect electrophysiological activity. Although blood oxygenation level-dependent (BOLD) fMRI has been widely used to map brain responses to external stimuli and to delineate functional circuits at rest, the extent to which BOLD signals correlate spatially with underlying neuronal activity, the spatial relationships between stimulus-evoked BOLD activations and local correlations of BOLD signals in a resting state, and whether these spatial relationships vary across functionally distinct cortical areas are not known. To address these critical questions, we directly compared the spatial extents of stimulated activations and the local profiles of intervoxel resting state correlations for both high-resolution BOLD at 9.4 T and local field potentials (LFPs), using 98-channel microelectrode arrays, in functionally distinct primary somatosensory areas 3b and 1 in nonhuman primates. Anatomic images of LFP and BOLD were coregistered within 0.10 mm accuracy. We found that the point spread functions (PSFs) of BOLD and LFP responses were comparable in the stimulus condition, and both estimates of activations were slightly more spatially constrained than local correlations at rest. The magnitudes of stimulus responses in area 3b were stronger than those in area 1 and extended in a medial to lateral direction. In addition, the reproducibility and stability of stimulus-evoked activation locations within and across both modalities were robust. Our work suggests that the intrinsic resolution of BOLD is not a limiting feature in practice and approaches the intrinsic precision achievable by multielectrode electrophysiology.

Correlated Disruption of Resting-State fMRI, LFP, and Spike Connectivity between Area 3b and S2 following Spinal Cord Injury in Monkeys

This study aims to understand how functional connectivity (FC) between areas 3b and S2 alters following input deprivation and the neuronal basis of disrupted FC of resting-state fMRI signals. We combined submillimeter fMRI with microelectrode recordings to localize the deafferented digit regions in areas 3b and S2 by mapping tactile stimulus-evoked fMRI activations before and after cervical dorsal column lesion in each male monkey. An average afferent disruption of 97% significantly reduced fMRI, local field potential (LFP), and spike responses to stimuli in both areas. Analysis of resting-state fMRI signal correlation, LFP coherence, and spike cross-correlation revealed significantly reduced functional connectivity between deafferented areas 3b and S2. The degrees of reductions in stimulus responsiveness and FC after deafferentation differed across fMRI, LFP, and spiking signals. The reduction of FC was much weaker than that of stimulus-evoked responses. Whereas the largest stimulus-evoked signal drop (∼80%) was observed in LFP signals, the greatest FC reduction was detected in the spiking activity (∼30%). fMRI signals showed mild reductions in stimulus responsiveness (∼25%) and FC (∼20%). The overall deafferentation-induced changes were quite similar in areas 3b and S2 across signals. Here we demonstrated that FC strength between areas 3b and S2 was much weakened by dorsal column lesion, and stimulus response reduction and FC disruption in fMRI covary with those of LFP and spiking signals in deafferented areas 3b and S2. These findings have important implications for fMRI studies aiming to probe FC alterations in pathological conditions involving deafferentation in humans. SIGNIFICANCE STATEMENT By directly comparing fMRI, local field potential, and spike signals in both tactile stimulation and resting states before and after severe disruption of dorsal column afferent, we demonstrated that reduction in fMRI responses to stimuli is accompanied by weakened resting-state fMRI functional connectivity (FC) in input-deprived and reorganized digit regions in area 3b of the S1 and S2. Concurrent reductions in local field potential and spike FC validated the use of resting-state fMRI signals for probing neural intrinsic FC alterations in pathological deafferented cortex, and indicated that disrupted FC between mesoscale functionally highly related regions may contribute to the behavioral impairments.

High-resolution functional MRI identified distinct global intrinsic functional networks of nociceptive posterior insula and S2 regions in squirrel monkey brain

ABSTRACT Numerous functional imaging and electrophysiological studies in humans and animals indicate that the two contiguous areas of secondary somatosensory cortex (S2) and posterior insula (pIns) are core regions in nociceptive processing and pain perception. In this study, we tested the hypothesis that the S2‐pIns connection serves as a hub for connecting distinct sensory and affective nociceptive processing networks in the squirrel monkey brain. At 9.4 T, we first mapped the brain regions that respond to nociceptive heat stimuli with high‐resolution fMRI, and then used seed‐based resting‐state fMRI (rsfMRI) analysis to delineate and refine the global intrinsic functional connectivity circuits of the proximal S2 and pIns regions. In each subject, nociceptive (47.5 °C) heat‐evoked fMRI activations were detected in many brain regions, including primary somatosensory (S1), S2, pIns, area 7b, anterior cingulate cortex (ACC), primary motor cortex, prefrontal cortex, supplementary motor area, thalamus, and caudate. Using the heat‐evoked fMRI activation foci in S2 and pIns as the seeds, voxel‐wise whole‐brain resting‐state functional connectivity (rsFC) analysis revealed strong functional connections between contralateral S2 and pIns, as well as their corresponding regions in the ipsilateral hemisphere. Spatial similarity and overlap analysis identified each region as part of two distinct intrinsic functional networks with 7% overlap: sensory S2‐S1‐area 7b and affective pIns‐ACC‐PCC networks. Moreover, a high degree of overlap was observed between the combined rsFC maps of nociceptive S2 and pIns regions and the nociceptive heat‐evoked activation map. In summary, our study provides evidence for the existence of two distinct intrinsic functional networks for S2 and pIns nociceptive regions, and these two networks are joined via the S2‐pIns connection. Brain regions that are involved in processing nociceptive inputs are also highly interconnected at rest. The presence of robust and distinct S1‐S2‐area 7b and pIns‐ACC‐PCC rsFC networks under anesthesia underscores their fundamental roles in processing nociceptive information. HIGHLIGHTSNociceptive heat evoked fMRI activations in multiple refined brain regions.Nociceptive S2 and pIns regions exhibited distinct global rsFC networks.S1‐S2‐area 7b and pIns‐ACC‐PCC rsFC networks joined via S2‐pIns connection.

Altered Spatiotemporal Dynamics of Cortical Activation to Tactile Stimuli in Somatosensory Area 3b and Area 1 of Monkeys after Spinal Cord Injury

Abstract Reactivation of deafferented cortex plays a key role in mediating the recovery of lost functions, although the precise mechanism is not fully understood. This study simultaneously characterized the dynamic spatiotemporal features of tactile responses in areas 3b and 1 before and 6–8 weeks after partial dorsal column lesion (DCL), and examined how the reactivation relates to the recovery of simple hand use in squirrel monkeys. A combination of high spatiotemporal resolution functional intrinsic optical imaging, microelectrode mapping, behavioral assessment, and tracer histology methods were used. Compared with the normal cortex, we found that the responses of deafferented areas 3b and 1 to 3 s of continuous 8 Hz tactile stimulation of a single digit were significantly weaker and more transient. This finding indicates a loss of response to sustained tactile stimuli. The activation area enlarged for areas 3b and 1 in both directions along digit representation (medial–lateral) and across areas (anterior–posterior). All subjects showed behavioral deficits in a food reaching-grasping-retrieving task within the first 5 weeks after DCL, but recovered at the time when optical images were acquired. Summarily, we showed that these populations of cortical neurons responded to peripheral tactile inputs, albeit in significantly altered manners in each area, several weeks after deafferentation. We propose that compromised ascending driven inputs, impaired lateral inhibition, and local integration of input signals may account for the altered spatiotemporal dynamics of the reactivated areas 3b and 1 cortices. Further investigation with large sample sizes is needed to fully characterize the effects of deafferentation on area 1 activation size.