AbstractNeuropeptides are abundant signaling molecules in the central nervous system. Yet remarkably little is known about their spatiotemporal spread and biological activity. Here, we developed an integrated optical approach using Plasmonic nAnovesicles and cell‐based neurotransmitter fluorescent engineered reporter (CNiFER), or PACE, to probe neuropeptide signaling in the mouse neocortex. Small volumes (fL to pL) of exogenously supplied somatostatin‐14 (SST) can be rapidly released under near‐infrared light stimulation from nanovesicles implanted in the brain and detected by SST2 CNiFERs with nM sensitivity. Our measurements reveal reduced but synchronized SST transmission within 130 μm, and markedly smaller and delayed transmission at longer distances. These measurements enabled a quantitative estimation of the SST loss rate due to peptide degradation and binding. PACE offers a new tool for determining the spatiotemporal scales of neuropeptide volume transmission and signaling in the brain.

Recent developments in optical microscopy, applicable for large-scale and longitudinal imaging of cortical activity in behaving animals, open unprecedented opportunities to gain a deeper understanding of neurovascular and neurometabolic coupling during different brain states. Future studies will leverage these tools to deliver foundational knowledge about brain state-dependent regulation of cerebral blood flow and metabolism, as well as regulation as a function of brain maturation and aging. This knowledge is of critical importance to interpret hemodynamic signals observed with functional magnetic resonance imaging (fMRI).

Functional connectivity, which reflects the spatial and temporal organization of intrinsic activity throughout the brain, is one of the most studied measures in human neuroimaging research. The noninvasive acquisition of resting state functional magnetic resonance imaging (rs-fMRI) allows the characterization of features designated as functional networks, functional connectivity gradients, and time-varying activity patterns that provide insight into the intrinsic functional organization of the brain and potential alterations related to brain dysfunction. Functional connectivity, hence, captures dimensions of the brain's activity that have enormous potential for both clinical and preclinical research. However, the mechanisms underlying functional connectivity have yet to be fully characterized, hindering interpretation of rs-fMRI studies. As in other branches of neuroscience, the identification of the neurophysiological processes that contribute to functional connectivity largely depends on research conducted on laboratory animals, which provide a platform where specific, multi-dimensional investigations that involve invasive measurements can be carried out. These highly controlled experiments facilitate the interpretation of the temporal correlations observed across the brain. Indeed, information obtained from animal experimentation to date is the basis for our current understanding of the underlying basis for functional brain connectivity. This review presents a compendium of some of the most critical advances in the field based on the efforts made by the animal neuroimaging community.

The blood-brain barrier (BBB) is highly selective and acts as the interface between the central nervous system and circulation. While the BBB is critical for maintaining brain homeostasis, it represents a formidable challenge for drug delivery. Here we synthesized gold nanoparticles (AuNPs) for targeting the tight junction specifically and demonstrated that transcranial picosecond laser stimulation of these AuNPs post intravenous injection increases the BBB permeability. The BBB permeability change can be graded by laser intensity, is entirely reversible, and involves increased paracellular diffusion. BBB modulation does not lead to significant disruption in the spontaneous vasomotion or the structure of the neurovascular unit. This strategy allows the entry of immunoglobulins and viral gene therapy vectors, as well as cargo-laden liposomes. We anticipate this nanotechnology to be useful for tissue regions that are accessible to light or fiberoptic application and to open new avenues for drug screening and therapeutic interventions in the central nervous system.

Neuropeptides are essential signaling molecules in the nervous system involved in modulating neural circuits and behavior. Although hypothesized to signal via volume transmission through G-protein coupled receptors (GPCR), remarkably little is known about their extrasynaptic diffusion. Here, we developed an all-optical approach to probe neuropeptide volume transmission in mouse neocortex. To control neuropeptide release, we engineered photosensitive nanovesicles with somatostatin-14 (SST) that is released with near-infrared light stimulation. To detect SST, we created a new cell-based neurotransmitter fluorescent engineered reporter (CNiFER) using the SST2 GPCR. Under two-photon imaging, we determined the time to activate SST2R at defined distances as well as the maximal distance and loss rate for SST volume transmission in neocortex. Importantly, we determined that SST transmission is significantly faster in neocortex with a chemically degraded extracellular matrix, a diseased condition indicated in neuroinflammation and Parkinson’s disease. These new neurotechnologies can reveal important biological signaling processes previously not possible, and provide new opportunities to investigate volume transmission in the brain.

Ultra-slow,-0.1-Hz variations in the oxygenation level of brain blood are widely used as an fMRI-based surrogate of "resting-state"neuronal activity. The temporal correlations among these fluctuations across the brain are interpreted as "functional connections"for maps and neurological diagnostics. Ultra-slow variations in oxygenation follow a cascade. First, they closely track changes in arteriole diameter. Second, interpretable functional connections arise when the ultra-slow changes in amplitude of gamma-band neuronal oscillations, which are shared across even far-flung but synaptically connected brain regions, entrain the-0.1-Hz vasomotor oscillation in diameter of local arterioles. Significant confounds to estimates of functional connectivity arise from residual vasomotor activity as well as arteriole dynamics driven by self-generated movements and subcortical common modulatory inputs. Last, methodological limitations of fMRI can lead to spurious functional connections. The neuronal generator of ultra-slow variations in gamma-band amplitude, including that associated with self-generated movements, remains an open issue.

Ultra-slow,-0.1-Hz variations in the oxygenation level of brain blood are widely used as an fMRI-based surrogate of "resting-state"neuronal activity. The temporal correlations among these fluctuations across the brain are interpreted as "functional connections"for maps and neurological diagnostics. Ultra-slow variations in oxygenation follow a cascade. First, they closely track changes in arteriole diameter. Second, interpretable functional connections arise when the ultra-slow changes in amplitude of gamma-band neuronal oscillations, which are shared across even far-flung but synaptically connected brain regions, entrain the-0.1-Hz vasomotor oscillation in diameter of local arterioles. Significant confounds to estimates of functional connectivity arise from residual vasomotor activity as well as arteriole dynamics driven by self-generated movements and subcortical common modulatory inputs. Last, methodological limitations of fMRI can lead to spurious functional connections. The neuronal generator of ultra-slow variations in gamma-band amplitude, including that associated with self-generated movements, remains an open issue.

The blood-brain barrier (BBB) tightly regulates the entry of molecules into the brain by tight junctions that seals the paracellular space and receptor-mediated transcytosis. It remains elusive to selectively modulate these mechanisms and to overcome BBB without significant neurotoxicity. Here we report that light stimulation of tight junction-targeted plasmonic nanoparticles selectively opens up the paracellular route to allow diffusion through the compromised tight junction and into the brain parenchyma. The BBB modulation does not impair vascular dynamics and associated neurovascular coupling, or cause significant neural injury. It further allows antibody and adeno-associated virus delivery into local brain regions. This novel method offers the first evidence of selectively modulating BBB tight junctions and opens new avenues for therapeutic interventions in the central nervous system.One Sentence Summary Gentle stimulation of molecular-targeted nanoparticles selectively opens up the paracellular pathway and allows macromolecules and gene therapy vectors into the brain.### Competing Interest StatementA patent has been filed based on these findings. The patent was published on 12/19/2019 as WO 2019/241623 A1.

BACKGROUND: Functional magnetic resonance imaging (fMRI) in awake behaving mice is well positioned to bridge the detailed cellular-level view of brain activity, which has become available owing to recent advances in microscopic optical imaging and genetics, to the macroscopic scale of human noninvasive observables. However, though microscopic (e.g., two-photon imaging) studies in behaving mice have become a reality in many laboratories, awake mouse fMRI remains a challenge. Owing to variability in behavior among animals, performing all types of measurements within the same subject is highly desirable and can lead to higher scientific rigor. METHODS: We demonstrated blood oxygenation level-dependent fMRI in awake mice implanted with long-term cranial windows that allowed optical access for microscopic imaging modalities and optogenetic stimulation. We started with two-photon imaging of single-vessel diameter changes (n = 1). Next, we implemented intrinsic optical imaging of blood oxygenation and flow combined with laser speckle imaging of blood flow obtaining a mesoscopic picture of the hemodynamic response (n = 16). Then we obtained corresponding blood oxygenation level-dependent fMRI data (n = 5). All measurements could be performed in the same mice in response to identical sensory and optogenetic stimuli. RESULTS: The cranial window did not deteriorate the quality of fMRI and allowed alternation between imaging modalities in each subject. CONCLUSIONS: This report provides a proof of feasibility for multiscale imaging approaches in awake mice. In the future, this protocol could be extended to include complex cognitive behaviors translatable to humans, such as sensory discrimination or attention.

Michèle Desjardins, Kıvılcım Kılıç, Martin Thunemann, Celine Mateo, Dominic Holland, Christopher G.L. Ferri, Jonathan A. Cremonesi, Baoqiang Li, Qun Cheng, Kimberly L. Weldy, Payam A. Saisan, David Kleinfeld, Takaki Komiyama, Thomas T. Liu, Robert Bussell, Eric C. Wong, Miriam Scadeng, Andrew K. Dunn, David A. Boas, Sava Sakadžić, Joseph B. Mandeville, Richard B. Buxton, Anders M. Dale, Anna Devor

I will discuss recent two-photon optical and high-field functional MRI measurements that link brain activity with changes in cortical arteriole and venule diameter. The data support a “Bagpipe” model (Drew et al PNAS 2011) in which arterioles dilate upon heightened intrinsic or stimulus-driven neuronal activity and form a reservoir of blood to perfuse cortex. Implications and extensions of this result will be discussed.

Resting-state signals in blood-oxygenation-level-dependent (BOLD) imaging are used to parcellate brain regions and define "functional connections" between regions. Yet a physiological link between fluctuations in blood oxygenation with those in neuronal signaling pathways is missing. We present evidence from studies on mouse cortex that modulation of vasomotion, i.e., intrinsic ultra-slow (0.1 Hz) fluctuations in arteriole diameter, provides this link. First, ultra-slow fluctuations in neuronal signaling, which occur as an envelope over γ-band activity, entrains vasomotion. Second, optogenetic manipulations confirm that entrainment is unidirectional. Third, co-fluctuations in the diameter of pairs of arterioles within the same hemisphere diminish to chance for separations >1.4 mm. Yet the diameters of arterioles in distant (>5 mm), mirrored transhemispheric sites strongly co-fluctuate; these correlations are diminished in acallosal mice. Fourth, fluctuations in arteriole diameter coherently drive fluctuations in blood oxygenation. Thus, entrainment of vasomotion links neuronal pathways to functional connections.

The spatial organization of mouse frontal cortex is poorly understood. Here, we used voltage-sensitive dye to image electrical activity in the dorsal cortex of awake head-restrained mice. Whisker-deflection evoked the earliest sensory response in a localized region of primary somatosensory cortex and visual stimulation evoked the earliest responses in a localized region of primary visual cortex. Over the next milliseconds, the initial sensory response spread within the respective primary sensory cortex and into the surrounding higher order sensory cortices. In addition, secondary hotspots in the frontal cortex were evoked by whisker and visual stimulation, with the frontal hotspot for whisker deflection being more anterior and lateral compared to the frontal hotspot evoked by visual stimulation. Investigating axonal projections, we found that the somatosensory whisker cortex and the visual cortex directly innervated frontal cortex, with visual cortex axons innervating a region medial and posterior to the innervation from somatosensory cortex, consistent with the location of sensory responses in frontal cortex. In turn, the axonal outputs of these two frontal cortical areas innervate distinct regions of striatum, superior colliculus, and brainstem. Sensory input, therefore, appears to map onto modality-specific regions of frontal cortex, perhaps participating in distinct sensorimotor transformations, and directing distinct motor outputs.

The ability to form an accurate map of sensory input to the brain is an essential aspect of interpreting functional brain signals. Here, we consider the somatotopic map of vibrissa-based touch in the primary somatosensory (vS1) cortex of mice. The vibrissae are represented by a Manhattan-like grid of columnar structures that are separated by inter-digitating septa. The development, dynamics and plasticity of this organization is widely used as a model system. Yet, the exact anatomical position of this organization within the vS1 cortex varies between individual mice. Targeting of a particular column in vivo therefore requires prior mapping of the activated cortical region, for instance by imaging the evoked intrinsic optical signal (eIOS) during vibrissa stimulation. Here, we describe a procedure for constructing a complete somatotopic map of the vibrissa representation in the vS1 cortex using eIOS. This enables precise targeting of individual cortical columns. We found, using C57BL/6 mice, that although the precise location of the columnar field varies between animals, the relative spatial arrangement of the columns is highly preserved. This finding enables us to construct a canonical somatotopic map of the vibrissae in the vS1 cortex. In particular, the position of any column, in absolute anatomical coordinates, can be established with near certainty when the functional representations in the vS1 cortex for as few as two vibrissae have been mapped with eIOS.This article is part of the themed issue 'Interpreting BOLD: a dialogue between cognitive and cellular neuroscience'.

Identification of the cellular players and molecular messengers that communicate neuronal activity to the vasculature driving cerebral hemodynamics is important for (1) the basic understanding of cerebrovascular regulation and (2) interpretation of functional Magnetic Resonance Imaging (fMRI) signals. Using a combination of optogenetic stimulation and 2-photon imaging in mice, we demonstrate that selective activation of cortical excitation and inhibition elicits distinct vascular responses and identify the vasoconstrictive mechanism as Neuropeptide Y (NPY) acting on Y1 receptors. The latter implies that task-related negative Blood Oxygenation Level Dependent (BOLD) fMRI signals in the cerebral cortex under normal physiological conditions may be mainly driven by the NPY-positive inhibitory neurons. Further, the NPY-Y1 pathway may offer a potential therapeutic target in cerebrovascular disease.

We present a two-photon microscope that images the full extent of murine cortex with an objective-limited spatial resolution across an 8 mm by 10 mm field. The lateral resolution is approximately 1 µm and the maximum scan speed is 5 mm/ms. The scan pathway employs large diameter compound lenses to minimize aberrations and performs near theoretical limits. We demonstrate the special utility of the microscope by recording resting-state vasomotion across both hemispheres of the murine brain through a transcranial window and by imaging histological sections without the need to stitch.

We review the organizational principles of the cortical vasculature and the underlying patterns of blood flow under normal conditions and in response to occlusion of single vessels. The cortex is sourced by a two‐dimensional network of pial arterioles that feeds a three‐dimensional network of subsurface microvessels in close proximity to neurons and glia. Blood flow within the surface and subsurface networks is largely insensitive to occlusion of a single vessel within either network. However, the penetrating arterioles that connect the pial network to the subsurface network are bottlenecks to flow; occlusion of even a single penetrating arteriole results in the death of a 500 μm diameter cylinder of cortical tissue despite the potential for collateral flow through microvessels. This pattern of flow is consistent with that calculated from a full reconstruction of the angioarchitecture. Conceptually, collateral flow is insufficient to compensate for the occlusion of a penetrating arteriole because penetrating venules act as shunts of blood that flows through collaterals. Future directions that stem from the analysis of the angioarchitecture concern cellular‐level issues, in particular the regulation of blood flow within the subsurface microvascular network, and system‐level issues, in particular the role of penetrating arteriole occlusions in human cognitive impairment.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 μm in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 μm below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.