Two-photon imaging of excitatory and inhibitory neural response to infrared neural stimulation.

Significance: Pulsed infrared neural stimulation (INS, 1875 nm) is an emerging neurostimulation technology that delivers focal pulsed heat to activate functionally specific mesoscale networks and holds promise for clinical application. However, little is known about its effect on excitatory and inhibitory cell types in cerebral cortex. Aim: Estimates of summed population neuronal response time courses provide a potential basis for neural and hemodynamic signals described in other studies. Approach: Using two-photon calcium imaging in mouse somatosensory cortex, we have examined the effect of INS pulse train application on hSyn neurons and mDlx neurons tagged with GCaMP6s. Results: We find that, in anesthetized mice, each INS pulse train reliably induces robust response in hSyn neurons exhibiting positive going responses. Surprisingly, mDlx neurons exhibit negative going responses. Quantification using the index of correlation illustrates responses are reproducible, intensity-dependent, and focal. Also, a contralateral activation is observed when INS applied. Conclusions: In sum, the population of neurons stimulated by INS includes both hSyn and mDlx neurons; within a range of stimulation intensities, this leads to overall excitation in the stimulated population, leading to the previously observed activations at distant post-synaptic sites.

Brainwide mesoscale functional networks revealed by focal infrared neural stimulation of the amygdala.

The primate amygdala serves to evaluate emotional content of sensory inputs and modulate emotional and social behaviors; it modulates cognitive, multisensory and autonomic circuits predominantly via the basal (BA), lateral (LA), and central (CeA) nuclei, respectively. Based on recent electrophysiological evidence suggesting mesoscale (millimeters-scale) nature of intra-amygdala functional organization, we have investigated the connectivity of these nuclei using Infrared Neural Stimulation of single mesoscale sites coupled with mapping in ultrahigh field 7T functional Magnetic Resonance Imaging (INS-fMRI). Stimulation of multiple sites within amygdala of single individuals evoked 'mesoscale functional connectivity maps', allowing comparison of BA, LA and CeA connected brainwide networks. This revealed a mesoscale nature of connected sites, complementary spatial patterns of functional connectivity, and topographic relationships of nucleus-specific connections. Our data reveal a functional architecture of systematically organized brainwide networks mediating sensory, cognitive, and autonomic influences from the amygdala.

Mesoscale organization of ventral and dorsal visual pathways in macaque monkey revealed by 7T fMRI.

In human and nonhuman primate brains, columnar (mesoscale) organization has been demonstrated to underlie both lower and higher order aspects of visual information processing. Previous studies have focused on identifying functional preferences of mesoscale domains in specific areas; but there has been little understanding of how mesoscale domains may cooperatively respond to single visual stimuli across dorsal and ventral pathways. Here, we have developed ultrahigh-field 7 T fMRI methods to enable simultaneous mapping, in individual macaque monkeys, of response in both dorsal and ventral pathways to single simple color and motion stimuli. We provide the first evidence that anatomical V2 cytochrome oxidase-stained stripes are well aligned with fMRI maps of V2 stripes, settling a long-standing controversy. In the ventral pathway, a systematic array of paired color and luminance processing domains across V4 was revealed, suggesting a novel organization for surface information processing. In the dorsal pathway, in addition to high quality motion direction maps of MT, MST and V3A, alternating color and motion direction domains in V3 are revealed. As well, submillimeter motion domains were observed in peripheral LIPd and LIPv. In sum, our study provides a novel global snapshot of how mesoscale networks in the ventral and dorsal visual pathways form the organizational basis of visual objection recognition and vision for action.

Representation of conspecific vocalizations in amygdala of awake marmosets.

Human speech and animal vocalizations are important for social communication and animal survival. Neurons in the auditory pathway are responsive to a range of sounds, from elementary sound features to complex acoustic sounds. For social communication, responses to distinct patterns of vocalization are usually highly specific to an individual conspecific call, in some species. This includes the specificity of sound patterns and embedded biological information. We conducted single-unit recordings in the amygdala of awake marmosets and presented calls used in marmoset communication, calls of other species and calls from specific marmoset individuals. We found that some neurons (47/262) in the amygdala distinguished 'Phee' calls from vocalizations of other animals and other types of marmoset vocalizations. Interestingly, a subset of Phee-responsive neurons (22/47) also exhibited selectivity to one out of the three Phees from two different 'caller' marmosets. Our findings suggest that, while it has traditionally been considered the key structure in the limbic system, the amygdala also represents a critical stage of socially relevant auditory perceptual processing.

Mapping information flow between the inferotemporal and prefrontal cortices via neural oscillations in memory retrieval and maintenance.

Interaction between the inferotemporal (ITC) and prefrontal (PFC) cortices is critical for retrieving information from memory and maintaining it in working memory. Neural oscillations provide a mechanism for communication between brain regions. However, it remains unknown how information flow via neural oscillations is functionally organized in these cortices during these processes. In this study, we apply Granger causality analysis to electrocorticographic signals from both cortices of monkeys performing visual association tasks to map information flow. Our results reveal regions within the ITC where information flow to and from the PFC increases via specific frequency oscillations to form clusters during memory retrieval and maintenance. Theta-band information flow in both directions increases in similar regions in both cortices, suggesting reciprocal information exchange in those regions. These findings suggest that specific subregions function as nodes in the memory information-processing network between the ITC and the PFC.

A framework and resource for global collaboration in non-human primate neuroscience.

As science and technology evolve, there is an increasing need for promotion of international scientific exchange. Collaborations, while offering substantial opportunities for scientists and benefit to society, also present challenges for those working with animal models, such as non-human primates (NHPs). Diversity in regulation of animal research is sometimes mistaken for the absence of common international welfare standards. Here, the ethical and regulatory protocols for 13 countries that have guidelines in place for biomedical research involving NHPs were assessed with a focus on neuroscience. Review of the variability and similarity in trans-national NHP welfare regulations extended to countries in Asia, Europe and North America. A tabulated resource was established to advance solution-oriented discussions and scientific collaborations across borders. Our aim is to better inform the public and other stakeholders. Through cooperative efforts to identify and analyze information with reference to evidence-based discussion, the proposed key ingredients may help to shape and support a more informed, open framework. This framework and resource can be expanded further for biomedical research in other countries.

BMI 2.0: Toward a technological interface with brainwide networks.

The field of brain machine interface has long sought a technology for brainwide interaction. In this issue of Neuron, Kim et al.1 present a novel method for dynamic, patterned, and precise optogenetic stimulation of mouse cortex in ultra-high-field MRI that portends such an interface.

A 16-channel loop array for in vivo macaque whole-brain imaging at 7 T.

Combining multimodal approaches with functional magnetic resonance imaging (fMRI) has catapulted the research on brain circuitries of non-human primates (NHPs) into a new era. However, many studies are constrained by a lack of appropriate RF coils. In this study, a single loop transmit and 16-channel receive array coil was constructed for brain imaging of macaques at 7 Tesla (7 T). The 16 receive channels were mounted on a 3D-printed helmet-shaped form closely fitting the macaque head, with fourteen openings arranged for multimodal devices around the cortical regions. Coil performance was evaluated by quantifying and comparing signal-to-noise ratio (SNR) maps, noise correlations, g-factor maps and flip-angle maps with a 28-channel commercial knee coil. The in vivo results suggested that the macaque coil has higher SNR in cortical regions and better acceleration ability in parallel imaging, which may benefit revealing mesoscale organizations in the brain.

Design and application of a multimodality-compatible 1Tx/6Rx RF coil for monkey brain MRI at 7T.

OBJECTIVE: Blood-oxygen-level-dependent functional MRI allows to investigte neural activities and connectivity. While the non-human primate plays an essential role in neuroscience research, multimodal methods combining functional MRI with other neuroimaging and neuromodulation enable us to understand the brain network at multiple scales. APPROACH: In this study, a tight-fitting helmet-shape receive array with a single transmit loop for anesthetized macaque brain MRI at 7T was fabricated with four openings constructed in the coil housing to accommodate multimodal devices, and the coil performance was quantitatively evaluated and compared to a commercial knee coil. In addition, experiments over three macaques with infrared neural stimulation (INS), focused ultrasound stimulation (FUS), and transcranial direct current stimulation (tDCS) were conducted. MAIN RESULTS: The RF coil showed higher transmit efficiency, comparable homogeneity, improved SNR and enlarged signal coverage over the macaque brain. Infrared neural stimulation was applied to the amygdala in deep brain region, and activations in stimulation sites and connected sites were detected, with the connectivity consistent with anatomical information. Focused ultrasound stimulation was applied to the left visual cortex, and activations were acquired along the ultrasound traveling path, with all time course curves consistent with pre-designed paradigms. The existence of transcranial direct current stimulation electrodes brought no interference to the RF system, as evidenced through high-resolution MPRAGE structure images. SIGNIFICANCE: This pilot study reveals the feasibility for brain investigation at multiple spatiotemporal scales, which may advance our understanding in dynamic brain networks.

Single-microvessel occlusion produces lamina-specific microvascular flow vasodynamics and signs of neurodegenerative change.

Recent studies have highlighted the importance of understanding the architecture and function of microvasculature, and dysfunction of these microvessels may underlie neurodegenerative disease. Here, we utilize a high-precision ultrafast laser-induced photothrombosis (PLP) method to occlude single capillaries and then quantitatively study the effects on vasodynamics and surrounding neurons. Analysis of the microvascular architecture and hemodynamics after single-capillary occlusion reveals distinct changes upstream vs. downstream branches, which shows rapid regional flow redistribution and local downstream blood-brain barrier (BBB) leakage. Focal ischemia via capillary occlusions surrounding labeled target neurons induces dramatic and rapid lamina-specific changes in neuronal dendritic architecture. Further, we find that micro-occlusion at two different depths within the same vascular arbor results in distinct effects on flow profiles in layers 2/3 vs layer 4. The current results reveal laminar-scale regulation distinctions in microinfarct response and raise the possibility that relatively greater impacts on microvascular function contribute to cognitive decline in neurodegenerative disease.

Functional topography of pulvinar-visual cortex networks in macaques revealed by INS-fMRI.

The pulvinar in the macaque monkey contains three divisions: the medial pulvinar (PM), the lateral pulvinar (PL), and the inferior pulvinar (PI). Anatomical studies have shown that connections of PM are preferentially distributed to higher association areas, those of PL are biased toward the ventral visual pathway, and those of PI are biased with the dorsal visual pathway. To study functional connections of the pulvinar at mesoscale, we used a novel method called INS-fMRI (infrared neural stimulation and functional magnetic resonance imaging). This method permits studies and comparisons of multiple pulvinar networks within single animals. As previously revealed, stimulations of different sites in PL and PI produced topographically organized focal activations in visual areas V1, V2, and V3. In contrast, PM stimulation elicited little or diffuse response. The relative activations of areas V1, V2, V3A, V3d, V3v, V4, MT, and MST revealed that connections of PL are biased to ventral pathway areas, and those of PI are biased to dorsal areas. Different statistical values of activated blood-oxygen-level-dependent responses produced the same center of activation, indicating stability of connectivity; it also suggests possible dynamics of broad to focal responses from single stimulation sites. These results demonstrate that infrared neural stimulation-induced connectivity is largely consistent with previous anatomical connectivity studies, thereby demonstrating validity of our novel method. In addition, it suggests additional interpretations of functional connectivity to complement anatomical studies.

Infrared neural stimulation in human cerebral cortex.

BACKGROUND: Modulation of brain circuits by electrical stimulation has led to exciting and powerful therapies for diseases such as Parkinson's. Because human brain organization is based in mesoscale (millimeter-scale) functional nodes, having a method that can selectively target such nodes could enable more precise, functionally specific stimulation therapies. Infrared Neural Stimulation (INS) is an emerging stimulation technology that stimulates neural tissue via delivery of tiny heat pulses. In nonhuman primates, this optical method provides focal intensity-dependent stimulation of the brain without tissue damage. However, whether INS application to the human central nervous system (CNS) is similarly effective is unknown. OBJECTIVE: To examine the effectiveness of INS on human cerebral cortex in intraoperative setting and to evaluate INS damage threshholds. METHODS: Five epileptic subjects undergoing standard lobectomy for epilepsy consented to this study. Cortical response to INS was assessed by intrinsic signal optical imaging (OI, a method that detects changes in tissue reflectance due to neuronal activity). A custom integrated INS and OI system was developed specifically for short-duration INS and OI acquisition during surgical procedures. Single pulse trains of INS with intensities from 0.2 to 0.8 J/cm2 were delivered to the somatosensory cortex and responses were recorded via optical imaging. Following tissue resection, histological analysis was conducted to evaluate damage threshholds. RESULTS: As assessed by OI, and similar to results in monkeys, INS induced responses in human cortex were highly focal (millimeter sized) and led to relative suppression of nearby cortical sites. Intensity dependence was observed at both stimulated and functionally connected sites. Histological analysis of INS-stimulated human cortical tissue provided damage threshold estimates. CONCLUSION: This is the first study demonstrating application of INS to human CNS and shows feasibility for stimulating single cortical nodes and associated sites and provided INS damage threshold estimates for cortical tissue. Our results suggest that INS is a promising tool for stimulation of functionally selective mesoscale circuits in the human brain, and may lead to advances in the future of precision medicine.

Spatial frequency representation in V2 and V4 of macaque monkey.

Spatial frequency (SF) is an important attribute in the visual scene and is a defining feature of visual processing channels. However, there remain many unsolved questions about how extrastriate areas in primate visual cortex code this fundamental information. Here, using intrinsic signal optical imaging in visual areas of V2 and V4 of macaque monkeys, we quantify the relationship between SF maps and (1) visual topography and (2) color and orientation maps. We find that in orientation regions, low to high SF is mapped orthogonally to orientation; in color regions, which are reported to contain orthogonal axes of color and lightness, low SFs tend to be represented more frequently than high SFs. This supports a population-based SF fluctuation related to the 'color/orientation' organizations. We propose a generalized hypercolumn model across cortical areas, comprised of two orthogonal parameters with additional parameters.

Targeted Optical Neural Stimulation: A New Era for Personalized Medicine.

Targeted optical neural stimulation comprises infrared neural stimulation and optogenetics, which affect the nervous system through induced thermal transients and activation of light-sensitive proteins, respectively. The main advantage of this pair of optical tools is high functional selectivity, which conventional electrical stimulation lacks. Over the past 15 years, the mechanism, safety, and feasibility of optical stimulation techniques have undergone continuous investigation and development. When combined with other methods like optical imaging and high-field functional magnetic resonance imaging, the translation of optical stimulation to clinical practice adds high value. We review the theoretical foundations and current state of optical stimulation, with a particular focus on infrared neural stimulation as a potential bridge linking optical stimulation to personalized medicine.

Precision 1070 nm Ultrafast Laser-Induced Photothrombosis of Depth-Targeted Vessels In Vivo.

The cerebrovasculature plays an essential role in neurovascular and homeostatic functions in health and disease conditions. Many efforts have been made for developing vascular thrombosis methods to study vascular dysfunction in vivo, while technical challenges remain, such as accuracy and depth-selectivity to target a single vessel in the cerebral cortex. Herein, this paper first demonstrates the evaluation and quantification of the feasibility and effects of Rose Bengal (RB)-induced photothrombosis with 720-1070 nm ultrafast lasers in a raster scan. A flexible and reproducible approach is then proposed to employ a 1070 nm ultrafast laser with a spiral scan for producing RB-induced occlusion, which is described as precision ultrafast laser-induced photothrombosis (PLP). Combine with two-photon microscopy imaging, this PLP displays highly precise and fast occlusion induction of various vessel types, sizes, and depths, which enhances the precision and power of the photothrombosis protocol. Overall, the PLP method provides a real-time, practical, precise, and depth-selected single-vessel photothrombosis technology in the cerebral cortex with commercially available optical equipment, which is crucial for exploring brain vascular function with high spatial-temporal resolution in the brain.

Large-depth three-photon fluorescence microscopy imaging of cortical microvasculature on nonhuman primates with bright AIE probe In vivo.

Multiphoton microscopy has been a powerful tool in brain research, three-photon fluorescence microscopy is increasingly becoming an emerging technique for neurological research of the cortex in depth. Nonhuman primates play important roles in the study of brain science because of their neural and vascular similarity to humans. However, there are few research results of three-photon fluorescence microscopy on the brain of nonhuman primates due to the lack of optimized imaging systems and excellent fluorescent probes. Here we introduced a bright aggregation-induced emission (AIE) probe with excellent three-photon fluorescence efficiency as well as facile synthesis process and we validated its biocompatibility in the macaque monkey. We achieved a large-depth vascular imaging of approximately 1 mm in the cerebral cortex of macaque monkey with our lab-modified three-photon fluorescence microscopy system and the AIE probe. Functional measurement of blood velocity in deep cortex capillaries was also performed. Furthermore, the comparison of cortical deep vascular structure parameters across species was presented on the monkey and mouse cortex. This work is the first in vivo three-photon fluorescence microscopic imaging research on the macaque monkey cortex reaching the imaging depth of ∼1 mm with the bright AIE probe. The results demonstrate the potential of three-photon microscopy as primate-compatible method for imaging fine vascular networks and will advance our understanding of vascular function in normal and disease in humans.

Representation of Cone-Opponent Color Space in Macaque Early Visual Cortices.

In primate vision, the encoding of color perception arises from three types of retinal cone cells (L, M, and S cones). The inputs from these cones are linearly integrated into two cone-opponent channels (cardinal axes) before the lateral geniculate nucleus. In subsequent visual cortical stages, color-preferring neurons cluster into functional domains within "blobs" in V1, "thin/color stripes" in V2, and "color bands" in V4. Here, we hypothesize that, with increasing cortical hierarchy, the functional organization of hue representation becomes more balanced and less dependent on cone opponency. To address this question, we used intrinsic signal optical imaging in macaque V1, V2, and V4 cortices to examine the domain-based representation of specific hues (here referred to as "hue domains") in cone-opponent color space (4 cardinal and 4 intermediate hues). Interestingly, we found that in V1, the relative size of S-cone hue preference domain was significantly smaller than that for other hues. This notable difference was less prominent in V2, and, in V4 was virtually absent, resulting in a more balanced representation of hues. In V2, hue clusters contained sequences of shifting preference, while in V4 the organization of hue clusters was more complex. Pattern classification analysis of these hue maps showed that accuracy of hue classification improved from V1 to V2 to V4. These results suggest that hue representation by domains in the early cortical hierarchy reflects a transformation away from cone-opponency and toward a full-coverage representation of hue.

Functionally specific and sparse domain-based micro-networks in monkey V1 and V2.

The cerebral cortices of human and nonhuman primate brains are characterized by submillimeter functional domains. However, little is known about the connections of single functional domains. Here, in macaque monkey visual cortex, we have developed a targeted focal electrical stimulation method, coupled with functional optical imaging, to map cortical networks with submillimeter precision in vivo. We find that single functional domains are a part of highly specific and sparse intra-areal and inter-areal micro-networks. Across color-related and orientation-related functionalities, these micro-networks exhibit parallel connection patterns, suggesting a common domain-based architecture. Moreover, these micro-networks shift topographically at a submillimeter scale, suggesting that they serve as a fundamental unit for cortical information processing. Our findings establish a domain-based connectional architecture in the primate brain and present new constraints for cortical map representation.