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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.

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.

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., Understanding mechanisms of cerebral oxygen regulation is critical for healthy brain function. Here the authors show that respiration is a key modulator of cerebral oxygenation, which will be helpful in better resolving neurally-generated functional brain imaging signals, such as BOLD fMRI.

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., Brain hemodynamic signals show "1/f-like" dynamics, but their relationship to neural activity is unclear. Using experimental and computational approaches, this study reveals that 1/f-like dynamics in brain oxygenation do not reflect neuronal activity, but instead may be explained by heterogeneity in red blood cells.

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.

Changes in cortical neural activity are coupled to changes in local arterial diameter and blood flow. However, the neuronal types and the signaling mechanisms that control the basal diameter of cerebral arteries or their evoked dilations are not well understood. Using chronic two-photon microscopy, electrophysiology, chemogenetics, and pharmacology in awake, head-fixed mice, we dissected the cellular mechanisms controlling the basal diameter and evoked dilation in cortical arteries. We found that modulation of overall neural activity up or down caused corresponding increases or decreases in basal arterial diameter. Surprisingly, modulation of pyramidal neuron activity had minimal effects on basal or evoked arterial dilation. Instead, the neurally-mediated component of arterial dilation was largely regulated through nitric oxide released by neuronal nitric oxide synthase (nNOS)-expressing neurons, whose activity was not reflected in electrophysiological measures of population activity. Our results show that cortical hemodynamic signals are not controlled by the average activity of the neural population, but rather the activity of a small ‘oligarchy’ of neurons.

A Drosophila model system is used to show that the hereditary spastic paraplegia proteins spastin and atlastin help axons but not dendrites regenerate. The endoplasmic reticulum concentrates at tips of regenerating axons but not dendrites, and this depends on spastin and atlastin., 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.

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., Author Summary Unlike many other cell types, most neurons last a lifetime. When injured, these cells often activate survival and repair strategies rather than dying. One such response is regeneration of the axon after it is injured. Axon regeneration is a conserved process activated by the same signaling cascade in worms, flies and mammals. Surprisingly we find that this signaling cascade first initiates a different response. This first response stabilizes the cell, and its downregulation by mitochondrial fission and caspases allows for maximum regeneration at later times. We propose that neurons respond to axon injury in a multi-step process with an early lock-down phase in which the cell is stabilized, followed by a more plastic state in which regeneration is maximized.

SummaryAxon 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.

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.