Low-intensity pulsed ultrasound stimulation (LIPUS) modulates microglial activation following intracortical microelectrode implantation.

Microglia are important players in surveillance and repair of the brain. Implanting an electrode into the cortex activates microglia, produces an inflammatory cascade, triggers the foreign body response, and opens the blood-brain barrier. These changes can impede intracortical brain-computer interfaces performance. Using two-photon imaging of implanted microelectrodes, we test the hypothesis that low-intensity pulsed ultrasound stimulation can reduce microglia-mediated neuroinflammation following the implantation of microelectrodes. In the first week of treatment, we found that low-intensity pulsed ultrasound stimulation increased microglia migration speed by 128%, enhanced microglia expansion area by 109%, and a reduction in microglial activation by 17%, indicating improved tissue healing and surveillance. Microglial coverage of the microelectrode was reduced by 50% and astrocytic scarring by 36% resulting in an increase in recording performance at chronic time. The data indicate that low-intensity pulsed ultrasound stimulation helps reduce the foreign body response around chronic intracortical microelectrodes.

Low-intensity pulsed ultrasound stimulation (LIPUS) modulates microglial activation following intracortical microelectrode implantation.

Microglia are important players in surveillance and repair of the brain. Their activation mediates neuroinflammation caused by intracortical microelectrode implantation, which impedes the application of intracortical brain-computer interfaces (BCIs). While low-intensity pulsed ultrasound stimulation (LIPUS) can attenuate microglial activation, its potential to modulate the microglia-mediated neuroinflammation and enhance the bio-integration of microelectrodes remains insufficiently explored. We found that LIPUS increased microglia migration speed from 0.59±0.04 to 1.35±0.07 µm/hr on day 1 and enhanced microglia expansion area from 44.50±6.86 to 93.15±8.77 µm 2 /min on day 7, indicating improved tissue healing and surveillance. Furthermore, LIPUS reduced microglial activation by 17% on day 6, vessel-associated microglia ratio from 70.67±6.15 to 40.43±3.87% on day 7, and vessel diameter by 20% on day 28. Additionally, microglial coverage of the microelectrode was reduced by 50% in week 1, indicating better tissue-microelectrode integration. These data reveal that LIPUS helps resolve neuroinflammation around chronic intracortical microelectrodes.

Arousal state transitions occlude sensory-evoked neurovascular coupling in neonatal mice.

In the adult sensory cortex, increases in neural activity elicited by sensory stimulation usually drive vasodilation mediated by neurovascular coupling. However, whether neurovascular coupling is the same in neonatal animals as adults is controversial, as both canonical and inverted responses have been observed. We investigated the nature of neurovascular coupling in unanesthetized neonatal mice using optical imaging, electrophysiology, and BOLD fMRI. We find in neonatal (postnatal day 15, P15) mice, sensory stimulation induces a small increase in blood volume/BOLD signal, often followed by a large decrease in blood volume. An examination of arousal state of the mice revealed that neonatal mice were asleep a substantial fraction of the time, and that stimulation caused the animal to awaken. As cortical blood volume is much higher during REM and NREM sleep than the awake state, awakening occludes any sensory-evoked neurovascular coupling. When neonatal mice are stimulated during an awake period, they showed relatively normal (but slowed) neurovascular coupling, showing that that the typically observed constriction is due to arousal state changes. These result show that sleep-related vascular changes dominate over any sensory-evoked changes, and hemodynamic measures need to be considered in the context of arousal state changes.

Arousal state transitions occlude sensory-evoked neurovascular coupling in neonatal mice.

In the adult sensory cortex, increases in neural activity elicited by sensory stimulation usually drives vasodilation mediated by neurovascular coupling. However, whether neurovascular coupling is the same in neonatal animals as adults is controversial, as both canonical and inverted responses have been observed. We investigated the nature of neurovascular coupling in unanesthetized neonatal mice using optical imaging, electrophysiology, and BOLD fMRI. We find in neonatal (postnatal day 15, P15) mice, sensory stimulation induces a small increase in blood volume/BOLD signal, often followed by a large decrease in blood volume. An examination of arousal state of the mice revealed that neonatal mice were asleep a substantial fraction of the time, and that stimulation caused the animal to awaken. As cortical blood volume is much higher during REM and NREM sleep than the awake state, awakening occludes any sensory-evoked neurovascular coupling. When neonatal mice are stimulated during an awake period, they showed relatively normal (but slowed) neurovascular coupling, showing that that the typically observed constriction is due to arousal state changes. These result show that sleep-related vascular changes dominate over any sensory-evoked changes, and hemodynamic measures need to be considered in the context of arousal state changes. Significance Statement: In the adult brain, increases in neural activity are often followed by vasodilation, allowing activity to be monitored using optical or magnetic resonance imaging. However, in neonates, sensory stimulation can drive vasoconstriction, whose origin was not understood. We used optical and magnetic resonance imaging approaches to investigate hemodynamics in neonatal mice. We found that sensory-induced vasoconstriction occurred when the mice were asleep, as sleep is associated with dilation of the vasculature of the brain relative to the awake state. The stimulus awakens the mice, causing a constriction due to the arousal state change. Our study shows the importance of monitoring arousal state, particularly when investigating subjects that may sleep, and the dominance arousal effects on brain hemodynamics.

Relating Pupil Diameter and Blinking to Cortical Activity and Hemodynamics across Arousal States.

Arousal state affects neural activity and vascular dynamics in the cortex, with sleep associated with large changes in the local field potential and increases in cortical blood flow. We investigated the relationship between pupil diameter and blink rate with neural activity and blood volume in the somatosensory cortex in male and female unanesthetized, head-fixed mice. We monitored these variables while the mice were awake, during periods of rapid eye movement (REM), and non-rapid eye movement (NREM) sleep. Pupil diameter was smaller during sleep than in the awake state. Changes in pupil diameter were coherent with both gamma-band power and blood volume in the somatosensory cortex, but the strength and sign of this relationship varied with arousal state. We observed a strong negative correlation between pupil diameter and both gamma-band power and blood volume during periods of awake rest and NREM sleep, although the correlations between pupil diameter and these signals became positive during periods of alertness, active whisking, and REM. Blinking was associated with increases in arousal and decreases in blood volume when the mouse was asleep. Bilateral coherence in gamma-band power and in blood volume dropped following awake blinking, indicating a reset of neural and vascular activity. Using only eye metrics (pupil diameter and eye motion), we could determine the arousal state of the mouse ('Awake,' 'NREM,' 'REM') with >90% accuracy with a 5 s resolution. There is a strong relationship between pupil diameter and hemodynamics signals in mice, reflecting the pronounced effects of arousal on cerebrovascular dynamics.SIGNIFICANCE STATEMENT Determining arousal state is a critical component of any neuroscience experiment. Pupil diameter and blinking are influenced by arousal state, as are hemodynamics signals in the cortex. We investigated the relationship between cortical hemodynamics and pupil diameter and found that pupil diameter was strongly related to the blood volume in the cortex. Mice were more likely to be awake after blinking than before, and blinking resets neural activity. Pupil diameter and eye motion can be used as a reliable, noninvasive indicator of arousal state. As mice transition from wake to sleep and back again over a timescale of seconds, monitoring pupil diameter and eye motion permits the noninvasive detection of sleep events during behavioral or resting-state experiments.

Behavioral and physiological monitoring for awake neurovascular coupling experiments: a how-to guide.

Significance: Functional brain imaging in awake animal models is a popular and powerful technique that allows the investigation of neurovascular coupling (NVC) under physiological conditions. However, ubiquitous facial and body motions (fidgeting) are prime drivers of spontaneous fluctuations in neural and hemodynamic signals. During periods without movement, animals can rapidly transition into sleep, and the hemodynamic signals tied to arousal state changes can be several times larger than sensory-evoked responses. Given the outsized influence of facial and body motions and arousal signals in neural and hemodynamic signals, it is imperative to detect and monitor these events in experiments with un-anesthetized animals. Aim: To cover the importance of monitoring behavioral state in imaging experiments using un-anesthetized rodents, and describe how to incorporate detailed behavioral and physiological measurements in imaging experiments. Approach: We review the effects of movements and sleep-related signals (heart rate, respiration rate, electromyography, intracranial pressure, whisking, and other body movements) on brain hemodynamics and electrophysiological signals, with a focus on head-fixed experimental setup. We summarize the measurement methods currently used in animal models for detection of those behaviors and arousal changes. We then provide a guide on how to incorporate this measurements with functional brain imaging and electrophysiology measurements. Results: We provide a how-to guide on monitoring and interpreting a variety of physiological signals and their applications to NVC experiments in awake behaving mice. Conclusion: This guide facilitates the application of neuroimaging in awake animal models and provides neuroscientists with a standard approach for monitoring behavior and other associated physiological parameters in head-fixed animals.

Awake mouse brain photoacoustic and optical imaging through a transparent ultrasound cranial window.

Optical resolution photoacoustic microscopy (OR-PAM) can map the cerebral vasculature at capillary-level resolution. However, the OR-PAM setup's bulky imaging head makes awake mouse brain imaging challenging and inhibits its integration with other optical neuroimaging modalities. Moreover, the glass cranial windows used for optical microscopy are unsuitable for OR-PAM due to the acoustic impedance mismatch between the glass plate and the tissue. To overcome these challenges, we propose a lithium niobate based transparent ultrasound transducer (TUT) as a cranial window on a thinned mouse skull. The TUT cranial window simplifies the imaging head considerably due to its dual functionality as an optical window and ultrasound transducer. The window remains stable for six weeks, with no noticeable inflammation and minimal bone regrowth. The TUT window's potential is demonstrated by imaging the awake mouse cerebral vasculature using OR-PAM, intrinsic optical signal imaging, and two-photon microscopy. The TUT cranial window can potentially also be used for ultrasound stimulation and simultaneous multimodal imaging of the awake mouse brain.

Brain-wide ongoing activity is responsible for significant cross-trial BOLD variability.

A notorious issue of task-based functional magnetic resonance imaging (fMRI) is its large cross-trial variability. To quantitatively characterize this variability, the blood oxygenation level-dependent (BOLD) signal can be modeled as a linear summation of a stimulation-relevant and an ongoing (i.e. stimulation-irrelevant) component. However, systematic investigation on the spatiotemporal features of the ongoing BOLD component and how these features affect the BOLD response is still lacking. Here we measured fMRI responses to light onsets and light offsets in awake rats. The neuronal response was simultaneously recorded with calcium-based fiber photometry. We established that between-region BOLD signals were highly correlated brain-wide at zero time lag, including regions that did not respond to visual stimulation, suggesting that the ongoing activity co-fluctuates across the brain. Removing this ongoing activity reduced cross-trial variability of the BOLD response by ~30% and increased its coherence with the Ca2+ signal. Additionally, the negative ongoing BOLD activity sometimes dominated over the stimulation-driven response and contributed to the post-stimulation BOLD undershoot. These results suggest that brain-wide ongoing activity is responsible for significant cross-trial BOLD variability, and this component can be reliably quantified and removed to improve the reliability of fMRI response. Importantly, this method can be generalized to virtually all fMRI experiments without changing stimulation paradigms.

Origins of 1/f-like tissue oxygenation fluctuations in the murine cortex.

The concentration of oxygen in the brain spontaneously fluctuates, and the distribution of power in these fluctuations has a 1/f-like spectra, where the power present at low frequencies of the power spectrum is orders of magnitude higher than at higher frequencies. Though these oscillations have been interpreted as being driven by neural activity, the origin of these 1/f-like oscillations is not well understood. Here, to gain insight of the origin of the 1/f-like oxygen fluctuations, we investigated the dynamics of tissue oxygenation and neural activity in awake behaving mice. We found that oxygen signal recorded from the cortex of mice had 1/f-like spectra. However, band-limited power in the local field potential did not show corresponding 1/f-like fluctuations. When local neural activity was suppressed, the 1/f-like fluctuations in oxygen concentration persisted. Two-photon measurements of erythrocyte spacing fluctuations and mathematical modeling show that stochastic fluctuations in erythrocyte flow could underlie 1/f-like dynamics in oxygenation. These results suggest that the discrete nature of erythrocytes and their irregular flow, rather than fluctuations in neural activity, could drive 1/f-like fluctuations in tissue oxygenation.

nNOS-expressing interneurons control basal and behaviorally evoked arterial dilation in somatosensory cortex of mice.

Cortical neural activity is coupled to local arterial diameter and blood flow. However, which neurons control the dynamics of cerebral arteries is not well understood. We dissected the cellular mechanisms controlling the basal diameter and evoked dilation in cortical arteries in awake, head-fixed mice. Locomotion drove robust arterial dilation, increases in gamma band power in the local field potential (LFP), and increases calcium signals in pyramidal and neuronal nitric oxide synthase (nNOS)-expressing neurons. Chemogenetic or pharmocological modulation of overall neural activity up or down caused corresponding increases or decreases in basal arterial diameter. Modulation of pyramidal neuron activity alone had little effect on basal or evoked arterial dilation, despite pronounced changes in the LFP. Modulation of the activity of nNOS-expressing neurons drove changes in the basal and evoked arterial diameter without corresponding changes in population neural activity.

Neurovascular coupling and bilateral connectivity during NREM and REM sleep.

To understand how arousal state impacts cerebral hemodynamics and neurovascular coupling, we monitored neural activity, behavior, and hemodynamic signals in un-anesthetized, head-fixed mice. Mice frequently fell asleep during imaging, and these sleep events were interspersed with periods of wake. During both NREM and REM sleep, mice showed large increases in cerebral blood volume ([HbT]) and arteriole diameter relative to the awake state, two to five times larger than those evoked by sensory stimulation. During NREM, the amplitude of bilateral low-frequency oscillations in [HbT] increased markedly, and coherency between neural activity and hemodynamic signals was higher than the awake resting and REM states. Bilateral correlations in neural activity and [HbT] were highest during NREM, and lowest in the awake state. Hemodynamic signals in the cortex are strongly modulated by arousal state, and changes during sleep are substantially larger than sensory-evoked responses.

Cerebral oxygenation during locomotion is modulated by respiration.

In the brain, increased neural activity is correlated with increases of cerebral blood flow and tissue oxygenation. However, how cerebral oxygen dynamics are controlled in the behaving animal remains unclear. We investigated to what extent cerebral oxygenation varies during locomotion. We measured oxygen levels in the cortex of awake, head-fixed mice during locomotion using polarography, spectroscopy, and two-photon phosphorescence lifetime measurements of oxygen sensors. We find that locomotion significantly and globally increases cerebral oxygenation, specifically in areas involved in locomotion, as well as in the frontal cortex and the olfactory bulb. The oxygenation increase persists when neural activity and functional hyperemia are blocked, occurred both in the tissue and in arteries feeding the brain, and is tightly correlated with respiration rate and the phase of respiration cycle. Thus, breathing rate is a key modulator of cerebral oxygenation and should be monitored during hemodynamic imaging, such as in BOLD fMRI.

Mitochondria and Caspases Tune Nmnat-Mediated Stabilization to Promote Axon Regeneration.

Axon injury can lead to several cell survival responses including increased stability and axon regeneration. Using an accessible Drosophila model system, we investigated the regulation of injury responses and their relationship. Axon injury stabilizes the rest of the cell, including the entire dendrite arbor. After axon injury we found mitochondrial fission in dendrites was upregulated, and that reducing fission increased stabilization or neuroprotection (NP). Thus axon injury seems to both turn on NP, but also dampen it by activating mitochondrial fission. We also identified caspases as negative regulators of axon injury-mediated NP, so mitochondrial fission could control NP through caspase activation. In addition to negative regulators of NP, we found that nicotinamide mononucleotide adenylyltransferase (Nmnat) is absolutely required for this type of NP. Increased microtubule dynamics, which has previously been associated with NP, required Nmnat. Indeed Nmnat overexpression was sufficient to induce NP and increase microtubule dynamics in the absence of axon injury. DLK, JNK and fos were also required for NP. Because NP occurs before axon regeneration, and NP seems to be actively downregulated, we tested whether excessive NP might inhibit regeneration. Indeed both Nmnat overexpression and caspase reduction reduced regeneration. In addition, overexpression of fos or JNK extended the timecourse of NP and dampened regeneration in a Nmnat-dependent manner. These data suggest that NP and regeneration are conflicting responses to axon injury, and that therapeutic strategies that boost NP may reduce regeneration.

Spastin, atlastin, and ER relocalization are involved in axon but not dendrite regeneration.

Mutations in >50 genes, including spastin and atlastin, lead to hereditary spastic paraplegia (HSP). We previously demonstrated that reduction of spastin leads to a deficit in axon regeneration in a Drosophila model. Axon regeneration was similarly impaired in neurons when HSP proteins atlastin, seipin, and spichthyin were reduced. Impaired regeneration was dependent on genetic background and was observed when partial reduction of HSP proteins was combined with expression of dominant-negative microtubule regulators, suggesting that HSP proteins work with microtubules to promote regeneration. Microtubule rearrangements triggered by axon injury were, however, normal in all genotypes. We examined other markers to identify additional changes associated with regeneration. Whereas mitochondria, endosomes, and ribosomes did not exhibit dramatic repatterning during regeneration, the endoplasmic reticulum (ER) was frequently concentrated near the tip of the growing axon. In atlastin RNAi and spastin mutant animals, ER accumulation near single growing axon tips was impaired. ER tip concentration was observed only during axon regeneration and not during dendrite regeneration. In addition, dendrite regeneration was unaffected by reduction of spastin or atlastin. We propose that the HSP proteins spastin and atlastin promote axon regeneration by coordinating concentration of the ER and microtubules at the growing axon tip.

Development of dendrite polarity in Drosophila neurons.

BACKGROUND: Drosophila neurons have dendrites that contain minus-end-out microtubules. This microtubule arrangement is different from that of cultured mammalian neurons, which have mixed polarity microtubules in dendrites. RESULTS: To determine whether Drosophila and mammalian dendrites have a common microtubule organization during development, we analyzed microtubule polarity in Drosophila dendritic arborization neuron dendrites at different stages of outgrowth from the cell body in vivo. As dendrites initially extended, they contained mixed polarity microtubules, like mammalian neurons developing in culture. Over a period of several days this mixed microtubule array gradually matured to a minus-end-out array. To determine whether features characteristic of dendrites were localized before uniform polarity was attained, we analyzed dendritic markers as dendrites developed. In all cases the markers took on their characteristic distribution while dendrites had mixed polarity. An axonal marker was also quite well excluded from dendrites throughout development, although this was perhaps more efficient in mature neurons. To confirm that dendrite character could be acquired in Drosophila while microtubules were mixed, we genetically disrupted uniform dendritic microtubule organization. Dendritic markers also localized correctly in this case. CONCLUSIONS: We conclude that developing Drosophila dendrites initially have mixed microtubule polarity. Over time they mature to uniform microtubule polarity. Dendrite identity is established before the mature microtubule arrangement is attained, during the period of mixed microtubule polarity.

Normal spastin gene dosage is specifically required for axon regeneration.

Axon regeneration allows neurons to repair circuits after trauma; however, most of the molecular players in this process remain to be identified. Given that microtubule rearrangements have been observed in injured neurons, we tested whether microtubule-severing proteins might play a role in axon regeneration. We found that axon regeneration is extremely sensitive to levels of the microtubule-severing protein spastin. Although microtubule behavior in uninjured neurons was not perturbed in animals heterozygous for a spastin null allele, axon regeneration was severely disrupted in this background. Two types of axon regeneration-regeneration of an axon from a dendrite after proximal axotomy and regeneration of an axon from the stump after distal axotomy-were defective in Drosophila with one mutant copy of the spastin gene. Other types of axon and dendrite outgrowth, including regrowth of dendrites after pruning, were normal in heterozygotes. We conclude that regenerative axon growth is uniquely sensitive to spastin gene dosage.