Contribution of Excitatory and Inhibitory Neuronal Activity to BOLD fMRI.

The BOLD fMRI response in the cortex is often assumed to reflect changes in excitatory neural activity. However, the contribution of inhibitory neurons to BOLD fMRI is unclear. Here, the role of inhibitory and excitatory activity was examined using multimodal approaches: electrophysiological recording, 15.2 T fMRI, optical intrinsic signal imaging, and modeling. Inhibitory and excitatory neuronal activity in the somatosensory cortex were selectively modulated by 20-s optogenetic stimulation of VGAT-ChR2 and CaMKII-ChR2 mice, respectively. Somatosensory stimulation and optogenetic stimulation of excitatory neurons induced positive BOLD responses in the somatosensory network, whereas stimulation of inhibitory neurons produced biphasic responses at the stimulation site, initial positive and later negative BOLD signals, and negative BOLD responses at downstream sites. When the stimulation duration was reduced to 5 s, the hemodynamic response of VGAT-ChR2 mice to optogenetic stimulation was only positive. Lastly, modeling performed from neuronal and hemodynamic data shows that the hemodynamic response function (HRF) of excitatory neurons is similar across different conditions, whereas the HRF of inhibitory neurons is highly sensitive to stimulation frequency and peaks earlier than that of excitatory neurons. Our study provides insights into the neurovascular coupling of excitatory and inhibitory neurons and the interpretation of BOLD fMRI signals.

Mitochondria modulate programmed neuritic retraction.

Neuritic retraction in the absence of overt neuronal death is a shared feature of normal aging and neurodegenerative disorders, but the intracellular mechanisms modulating this process are not understood. We propose that cumulative distal mitochondrial protein damage results in impaired protein import, leading to mitochondrial dysfunction and focal activation of the canonical apoptosis pathway in neurites. This is a controlled process that may not lead to neuronal death and, thus, we term this phenomenon "neuritosis." Consistent with our hypothesis, we show that in primary cerebrocortical neurons, mitochondrial distance from the soma correlates with increased mitochondrial protein damage, PINK1 accumulation, reactive oxygen species production, and decreased mitochondrial membrane potential and depolarization threshold. Furthermore, we demonstrate that the distance-dependent mitochondrial membrane potential gradient exists in vivo in mice. We demonstrate that impaired distal mitochondria have a lower threshold for focal/nonlethal neuritic caspase-3 activation in normal neurons that is exacerbated in aging, stress, and neurodegenerative conditions, thus delineating a fundamental mechanistic underpinning for synaptic vulnerability.

Postsynaptic activity of inhibitory neurons evokes hemodynamic fMRI responses.

Functional MRI responses are localized to the synaptic sites of evoked inhibitory neurons, but it is unknown whether, or by what mechanisms, these neurons initiate functional hyperemia. Here, the neuronal origins of these hemodynamic responses were investigated by fMRI or local field potential and blood flow measurements during topical application of pharmacological agents when GABAergic granule cells in the rat olfactory bulb were synaptically targeted. First, to examine if postsynaptic activation of these inhibitory neurons was required for neurovascular coupling, we applied an NMDA receptor antagonist during cerebral blood volume-weighted fMRI acquisition and found that responses below the drug application site (up to ~1.5 mm) significantly decreased within ~30 min. Similarly, large decreases in granule cell postsynaptic activities and blood flow responses were observed when AMPA or NMDA receptor antagonists were applied. Second, inhibition of nitric oxide synthase preferentially decreased the initial, fast component of the blood flow response, while inhibitors of astrocyte-specific glutamate transporters and vasoactive intestinal peptide receptors did not decrease blood flow responses. Third, inhibition of GABA release with a presynaptic GABAB receptor agonist caused less reduction of neuronal and blood flow responses compared to the postsynaptic glutamate receptor antagonists. In conclusion, local hyperemia by synaptically-evoked inhibitory neurons was primarily driven by their postsynaptic activities, possibly through NMDA receptor-dependent calcium signaling that was not wholly dependent on nitric oxide.

Calcium activation of cortical neurons by continuous electrical stimulation: Frequency dependence, temporal fidelity, and activation density.

Electrical stimulation of the brain has become a mainstay of fundamental neuroscience research and an increasingly prevalent clinical therapy. Despite decades of use in basic neuroscience research and the growing prevalence of neuromodulation therapies, gaps in knowledge regarding activation or inactivation of neural elements over time have limited its ability to adequately interpret evoked downstream responses or fine-tune stimulation parameters to focus on desired responses. In this work, in vivo two-photon microscopy was used to image neuronal calcium activity in layer 2/3 neurons of somatosensory cortex (S1) in male C57BL/6J-Tg(Thy1-GCaMP6s)GP4.3Dkim/J mice during 30 s of continuous electrical stimulation at varying frequencies. We show frequency-dependent differences in spatial and temporal somatic responses during continuous stimulation. Our results elucidate conflicting results from prior studies reporting either dense spherical activation of somas biased toward those near the electrode, or sparse activation of somas at a distance via axons near the electrode. These findings indicate that the neural element specific temporal response local to the stimulating electrode changes as a function of applied charge density and frequency. These temporal responses need to be considered to properly interpret downstream circuit responses or determining mechanisms of action in basic science experiments or clinical therapeutic applications.

Inhibitory Neuron Activity Contributions to Hemodynamic Responses and Metabolic Load Examined Using an Inhibitory Optogenetic Mouse Model.

Hemodynamic signals are routinely used to noninvasively assess brain function in humans and animals. This work examined the contribution of inhibitory neuron activity on hemodynamic responses captured by changes in blood flow, volume and oxygenation in the cortex of lightly anesthetized mice. Because cortical activity is not commonly initiated by inhibitory neurons, experiments were conducted to examine the neuronal activity properties elicited by photo-stimulation. We observed comparable increases in neuronal activity evoked by forelimb and photo-stimulation; however, significantly larger increases in blood flow and volume were produced by photo-stimulation of inhibitory neurons compared with forelimb stimulation. Following blockade of glutamate and GABA-A receptors to reduce postsynaptic activity contributions, neuronal activity was reliably modulated and hemodynamic changes persisted, though slightly reduced. More importantly, photo-stimulation-evoked changes in blood flow and volume were suppressed by 75-80% with the administration of a nitric oxide synthase inhibitor, suggesting that inhibitory neurons regulate blood flow mostly via nitric oxide. Lastly, forelimb and photo-stimulation of excitatory neurons produced local decreases in blood oxygenation, while large increases were generated by photo-stimulation of inhibitory neurons. Estimates of oxygen metabolism suggest that inhibitory neuron activity has a small impact on tissue metabolic load, indicating a mismatch between the metabolic demand and blood flow regulation properties of inhibitory and excitatory neurons.

Macroscale variation in resting-state neuronal activity and connectivity assessed by simultaneous calcium imaging, hemodynamic imaging and electrophysiology.

Functional imaging of spontaneous activity continues to play an important role in the field of connectomics. The most common imaging signal used for these experiments is the blood-oxygen-level-dependent (BOLD) functional MRI (fMRI) signal, but how this signal relates to spontaneous neuronal activity remains incompletely understood. Genetically encoded calcium indicators represent a promising tool to study this problem, as they can provide brain-wide measurements of neuronal activity compared to point measurements afforded by electrophysiological recordings. However, the relationship between the calcium signal and neurophysiological parameters at the mesoscopic scale requires further systematic characterization. Therefore, we collected simultaneous resting-state measurements of electrophysiology, along with calcium and hemodynamic imaging, in lightly anesthetized mice to investigate two aims. First, we examined the relationship between each imaging signal and the simultaneously recorded electrophysiological signal in a single brain region, finding that both signals are better correlated with multi-unit activity compared to local field potentials, with the calcium signal possessing greater signal-to-noise ratio and regional specificity. Second, we used the resting-state imaging data to model the relationship between the calcium and hemodynamic signals across the brain. We found that this relationship varied across brain regions in a way that is consistent across animals, with delays increasing by600 ms towards posterior cortical regions. Furthermore, while overall functional connectivity (FC) measured by the hemodynamic signal is significantly correlated with FC measured by calcium, the two estimates were found to be significantly different. We hypothesize that these differences arise at least in part from the observed regional variation in the hemodynamic response. In total, this work highlights some of the caveats needed in interpreting hemodynamic-based measurements of FC, as well as the need for improved modeling methods to reduce this potential source of bias.

A Materials Roadmap to Functional Neural Interface Design.

Advancement in neurotechnologies for electrophysiology, neurochemical sensing, neuromodulation, and optogenetics are revolutionizing scientific understanding of the brain while enabling treatments, cures, and preventative measures for a variety of neurological disorders. The grand challenge in neural interface engineering is to seamlessly integrate the interface between neurobiology and engineered technology, to record from and modulate neurons over chronic timescales. However, the biological inflammatory response to implants, neural degeneration, and long-term material stability diminish the quality of interface overtime. Recent advances in functional materials have been aimed at engineering solutions for chronic neural interfaces. Yet, the development and deployment of neural interfaces designed from novel materials have introduced new challenges that have largely avoided being addressed. Many engineering efforts that solely focus on optimizing individual probe design parameters, such as softness or flexibility, downplay critical multi-dimensional interactions between different physical properties of the device that contribute to overall performance and biocompatibility. Moreover, the use of these new materials present substantial new difficulties that must be addressed before regulatory approval for use in human patients will be achievable. In this review, the interdependence of different electrode components are highlighted to demonstrate the current materials-based challenges facing the field of neural interface engineering.

Enduring disturbances in regional cerebral blood flow and brain oxygenation at 24 h after asphyxial cardiac arrest in developing rats.

BACKGROUND: Disturbances in cerebral blood flow (CBF) and brain oxygenation (PbO2) are present early after pediatric cardiac arrest (CA). CBF-targeted therapies improved neurological outcome in our CA model. To assess the therapeutic window for CBF- and PbO2-targeted therapies, we propose to determine if CBF and PbO2 disturbances persist at 24 h after experimental pediatric CA. METHODS: Regional CBF and PbO2 were measured at 24 h after asphyxial CA in immature rats (n = 26, 6-8/group) using arterial spin label MRI and tissue electrodes, respectively. RESULTS: In all regions but the thalamus, CBF recovered to sham values by 24 h; thalamic CBF was >32% higher after CA vs. sham. PbO2 values at 24 h after CA in the cortex and thalamus were similar to shams in rats who received supplemental oxygen, however, on room air, cortical PbO2 was lower after CA vs. shams. CONCLUSION: CBF remains increased in the thalamus at 24 h after CA and PbO2 is decreased to hypoxic levels in cortex at 24 h after CA in rats who do not receive supplemental oxygen. Given the enduring disturbances in this model and the lack of routine CBF or PbO2 monitoring in patients, our data suggest the need for clinical correlation.

Improved spatial accuracy of functional maps in the rat olfactory bulb using supervised machine learning approach.

Functional MRI (fMRI) is a popular and important tool for noninvasive mapping of neural activity. As fMRI measures the hemodynamic response, the resulting activation maps do not perfectly reflect the underlying neural activity. The purpose of this work was to design a data-driven model to improve the spatial accuracy of fMRI maps in the rat olfactory bulb. This system is an ideal choice for this investigation since the bulb circuit is well characterized, allowing for an accurate definition of activity patterns in order to train the model. We generated models for both cerebral blood volume weighted (CBVw) and blood oxygen level dependent (BOLD) fMRI data. The results indicate that the spatial accuracy of the activation maps is either significantly improved or at worst not significantly different when using the learned models compared to a conventional general linear model approach, particularly for BOLD images and activity patterns involving deep layers of the bulb. Furthermore, the activation maps computed by CBVw and BOLD data show increased agreement when using the learned models, lending more confidence to their accuracy. The models presented here could have an immediate impact on studies of the olfactory bulb, but perhaps more importantly, demonstrate the potential for similar flexible, data-driven models to improve the quality of activation maps calculated using fMRI data.

Neural and hemodynamic responses elicited by forelimb- and photo-stimulation in channelrhodopsin-2 mice: insights into the hemodynamic point spread function.

Hemodynamic responses are commonly used to map brain activity; however, their spatial limits have remained unclear because of the lack of a well-defined and malleable spatial stimulus. To examine the properties of neural activity and hemodynamic responses, multiunit activity, local field potential, cerebral blood volume (CBV)-sensitive optical imaging, and laser Doppler flowmetry were measured from the somatosensory cortex of transgenic mice expressing Channelrhodopsin-2 in cortex Layer 5 pyramidal neurons. The magnitude and extent of neural and hemodynamic responses were modulated using different photo-stimulation parameters and compared with those induced by somatosensory stimulation. Photo-stimulation-evoked spiking activity across cortical layers was similar to forelimb stimulation, although their activity originated in different layers. Hemodynamic responses induced by forelimb- and photo-stimulation were similar in magnitude and shape, although the former were slightly larger in amplitude and wider in extent. Altogether, the neurovascular relationship differed between these 2 stimulation pathways, but photo-stimulation-evoked changes in neural and hemodynamic activities were linearly correlated. Hemodynamic point spread functions were estimated from the photo-stimulation data and its full-width at half-maximum ranged between 103 and 175 µm. Therefore, submillimeter functional structures separated by a few hundred micrometers may be resolved using hemodynamic methods, such as optical imaging and functional magnetic resonance imaging.

Revealing in vivo cellular mechanisms of cerebral microbleeds on neurons and microglia across cortical layers.

Cerebral microbleeds (CMBs) are associated with higher risk for various neurological diseases including stroke, dementia, and Alzheimer's disease. However, the understanding of cellular pathology of CMBs, particularly in deep brain regions, remains limited. Utilizing two-photon microscopy and microprism implantation, we longitudinally imaged the impact of CMBs on neuronal and microglial activities across cortical depths in awake mice. A temporary decline in spontaneous neuronal activity occurred throughout cortical layers, followed by recovery within a week. However, significant changes of neuron-neuron activity correlations persisted for weeks. Moreover, microglial contact with neuron soma significantly increased post-microbleeds, indicating an important modulatory role of microglia. Notably, microglial contact, negatively correlated with neuronal firing rate in normal conditions, became uncorrelated after microbleeds, suggesting a decreased neuron-microglia inhibition. These findings reveal chronic alterations in cortical neuronal networks and microglial-neuronal interactions across cortical depths, shedding light on the pathology of CMBs.

Activation and depression of neural and hemodynamic responses induced by the intracortical microstimulation and visual stimulation in the mouse visual cortex.

Objective. Intracortical microstimulation (ICMS) can be an effective method for restoring sensory perception in contemporary brain-machine interfaces. However, the mechanisms underlying better control of neuronal responses remain poorly understood, as well as the relationship between neuronal activity and other concomitant phenomena occurring around the stimulation site.Approach. Different microstimulation frequencies were investigatedin vivoon Thy1-GCaMP6s mice using widefield and two-photon imaging to evaluate the evoked excitatory neural responses across multiple spatial scales as well as the induced hemodynamic responses. Specifically, we quantified stimulation-induced neuronal activation and depression in the mouse visual cortex and measured hemodynamic oxyhemoglobin and deoxyhemoglobin signals using mesoscopic-scale widefield imaging.Main results. Our calcium imaging findings revealed a preference for lower-frequency stimulation in driving stronger neuronal activation. A depressive response following the neural activation preferred a slightly higher frequency stimulation compared to the activation. Hemodynamic signals exhibited a comparable spatial spread to neural calcium signals. Oxyhemoglobin concentration around the stimulation site remained elevated during the post-activation (depression) period. Somatic and neuropil calcium responses measured by two-photon microscopy showed similar dependence on stimulation parameters, although the magnitudes measured in soma was greater than in neuropil. Furthermore, higher-frequency stimulation induced a more pronounced activation in soma compared to neuropil, while depression was predominantly induced in soma irrespective of stimulation frequencies.Significance. These results suggest that the mechanism underlying depression differs from activation, requiring ample oxygen supply, and affecting neurons. Our findings provide a novel understanding of evoked excitatory neuronal activity induced by ICMS and offer insights into neuro-devices that utilize both activation and depression phenomena to achieve desired neural responses.

Activation and depression of neural and hemodynamic responses induced by the intracortical microstimulation and visual stimulation in the mouse visual cortex.

Objective . Intracortical microstimulation can be an effective method for restoring sensory perception in contemporary brain-machine interfaces. However, the mechanisms underlying better control of neuronal responses remain poorly understood, as well as the relationship between neuronal activity and other concomitant phenomena occurring around the stimulation site. Approach . Different microstimulation frequencies were investigated in vivo on Thy1-GCaMP6s mice using widefield and two-photon imaging to evaluate the evoked excitatory neural responses across multiple spatial scales as well as the induced hemodynamic responses. Specifically, we quantified stimulation-induced neuronal activation and depression in the mouse visual cortex and measured hemodynamic oxyhemoglobin and deoxyhemoglobin signals using mesoscopic-scale widefield imaging. Main results . Our calcium imaging findings revealed a preference for lower-frequency stimulation in driving stronger neuronal activation. A depressive response following the neural activation preferred a slightly higher frequency stimulation compared to the activation. Hemodynamic signals exhibited a comparable spatial spread to neural calcium signals. Oxyhemoglobin concentration around the stimulation site remained elevated during the post-activation (depression) period. Somatic and neuropil calcium responses measured by two-photon microscopy showed similar dependence on stimulation parameters, although the magnitudes measured in soma was greater than in neuropil. Furthermore, higher-frequency stimulation induced a more pronounced activation in soma compared to neuropil, while depression was predominantly induced in soma irrespective of stimulation frequencies. Significance . These results suggest that the mechanism underlying depression differs from activation, requiring ample oxygen supply, and affecting neurons. Our findings provide a novel understanding of evoked excitatory neuronal activity induced by intracortical microstimulation and offer insights into neuro-devices that utilize both activation and depression phenomena to achieve desired neural responses.

Electrically Controlled Vasodilator Delivery from PEDOT/Silica Nanoparticle Modulates Vessel Diameter in Mouse Brain.

Vascular damage and reduced tissue perfusion are expected to majorly contribute to the loss of neurons or neural signals around implanted electrodes. However, there are limited methods of controlling the vascular dynamics in tissues surrounding these implants. This work utilizes conducting polymer poly(ethylenedioxythiophene) and sulfonated silica nanoparticle composite (PEDOT/SNP) to load and release a vasodilator, sodium nitroprusside, to controllably dilate the vasculature around carbon fiber electrodes (CFEs) implanted in the mouse cortex. The vasodilator release is triggered via electrical stimulation and the amount of release increases with increasing electrical pulses. The vascular dynamics are monitored in real-time using two-photon microscopy, with changes in vessel diameters quantified before, during, and after the release of the vasodilator into the tissues. This work observes significant increases in vessel diameters when the vasodilator is electrically triggered to release, and differential effects of the drug release on vessels of different sizes. In conclusion, the use of nanoparticle reservoirs in conducting polymer-based drug delivery platforms enables the controlled delivery of vasodilator into the implant environment, effectively altering the local vascular dynamics on demand. With further optimization, this technology could be a powerful tool to improve the neural electrode-tissue interface and study neurovascular coupling.

Integrated Microprism and Microelectrode Array for Simultaneous Electrophysiology and Two-Photon Imaging across All Cortical Layers.

Cerebral neural electronics play a crucial role in neuroscience research with increasing translational applications such as brain-computer interfaces for sensory input and motor output restoration. While widely utilized for decades, the understanding of the cellular mechanisms underlying this technology remains limited. Although two-photon microscopy (TPM) has shown great promise in imaging superficial neural electrodes, its application to deep-penetrating electrodes is technically difficult. Here, a novel device integrating transparent microelectrode arrays with glass microprisms, enabling electrophysiology recording and stimulation alongside TPM imaging across all cortical layers in a vertical plane, is introduced. Tested in Thy1-GCaMP6 mice for over 4 months, the integrated device demonstrates the capability for multisite electrophysiological recording/stimulation and simultaneous TPM calcium imaging. As a proof of concept, the impact of microstimulation amplitude, frequency, and depth on neural activation patterns is investigated using the setup. With future improvements in material stability and single unit yield, this multimodal tool greatly expands integrated electrophysiology and optical imaging from the superficial brain to the entire cortical column, opening new avenues for neuroscience research and neurotechnology development.

Long-range inhibitory neurons mediate cortical neurovascular coupling.

To meet the high energy demands of brain function, cerebral blood flow (CBF) parallels changes in neuronal activity by a mechanism known as neurovascular coupling (NVC). However, which neurons play a role in mediating NVC is not well understood. Here, we identify in mice and humans a specific population of cortical GABAergic neurons that co-express neuronal nitric oxide synthase and tachykinin receptor 1 (Tacr1). Through whole-tissue clearing, we demonstrate that Tacr1 neurons extend local and long-range projections across functionally connected cortical areas. We show that whisker stimulation elicited Tacr1 neuron activity in the barrel cortex through feedforward excitatory pathways. Additionally, through optogenetic experiments, we demonstrate that Tacr1 neurons are instrumental in mediating CBF through the relaxation of mural cells in a similar fashion to whisker stimulation. Finally, by electron microscopy, we observe that Tacr1 processes contact astrocytic endfeet. These findings suggest that Tacr1 neurons integrate cortical activity to mediate NVC.

Effects of the α2-adrenergic receptor agonist dexmedetomidine on neural, vascular and BOLD fMRI responses in the somatosensory cortex.

This article describes the effects of dexmedetomidine (DEX) - the active ingredient of medetomidine, which is the latest popular sedative for functional magnetic resonance imaging (fMRI) in rodents - on multiple unit activity, local field potential (LFP), cerebral blood flow (CBF), pial vessel diameter [indicative of cerebral blood volume (CBV)], and blood oxygenation level-dependent (BOLD) fMRI. These measurements were obtained from the rat somatosensory cortex during 10 s of forepaw stimulation. We found that the continuous intravascular systemic infusion of DEX (50 µg/kg/h, doses typically used in fMRI studies) caused epileptic activities, and that supplemental isoflurane (ISO) administration of ~0.3% helped to suppress the development of epileptic activities and maintained robust neuronal and hemodynamic responses for up to 3 h. Supplemental administration of N(2)O in addition to DEX nearly abolished hemodynamic responses even if neuronal activity remained. Under DEX + ISO anesthesia, spike firing rate and the delta power of LFP increased, whereas beta and gamma power decreased, as compared with ISO-only anesthesia. DEX administration caused pial arteries and veins to constrict nearly equally, resulting in decreases in baseline CBF and CBV. Evoked LFP and CBF responses to forepaw stimulation were largest at a frequency of 8-10 Hz, and a non-linear relationship was observed. Similarly, BOLD fMRI responses measured at 9.4 T were largest at a frequency of 10 Hz. Both pial arteries and veins dilated rapidly (artery, 32.2%; vein, 5.8%), and venous diameter returned to baseline slower than arterial diameter. These results will be useful for designing, conducting and interpreting fMRI experiments under DEX sedation.

Resuscitation with epinephrine worsens cerebral capillary no-reflow after experimental pediatric cardiac arrest: An in vivo multiphoton microscopy evaluation.

Epinephrine is the principal resuscitation therapy for pediatric cardiac arrest (CA). Clinical data suggest that although epinephrine increases the rate of resuscitation, it fails to improve neurological outcome, possibly secondary to reductions in microvascular flow. We characterized the effect of epinephrine vs. placebo administered at resuscitation from pediatric asphyxial CA on microvascular and macrovascular cortical perfusion assessed using in vivo multiphoton microscopy and laser speckle flowmetry, respectively, and on brain tissue oxygenation (PbO2), behavioral outcomes, and neuropathology in 16-18-day-old rats. Epinephrine-treated rats had a more rapid return of spontaneous circulation and brisk immediate cortical reperfusion during 1-3 min post-CA vs. placebo. However, at the microvascular level, epinephrine-treated rats had penetrating arteriole constriction and increases in both capillary stalling (no-reflow) and cortical capillary transit time 30-60 min post-CA vs. placebo. Placebo-treated rats had increased capillary diameters post-CA. The cortex was hypoxic post-CA in both groups. Epinephrine treatment worsened reference memory performance vs. shams. Hippocampal neuron counts did not differ between groups. Resuscitation with epinephrine enhanced immediate reperfusion but produced microvascular alterations during the first hour post-resuscitation, characterized by vasoconstriction, capillary stasis, prolonged cortical transit time, and absence of compensatory cortical vasodilation. Targeted therapies mitigating the deleterious microvascular effects of epinephrine are needed.

Long-term in vivo two-photon imaging of the neuroinflammatory response to intracortical implants and micro-vessel disruptions in awake mice.

Our understanding of biomaterials in the brain have been greatly enhanced by advancements in in vivo imaging technologies such as two-photon microscopy. However, when applied to chronic studies, two-photon microscopy enables high-resolution imaging only in superficial regions due to inflammatory responses introduced by the craniotomy and insertion of foreign biomaterials. Microprisms provide a unique vertical view from brain surface to ~1 mm deep or more (depending on the size of the microprisms) which may break through this limitation on imaging depth. Although microprism has been used in the field of neuroscience, the in vivo foreign body responses to the microprism implant have yet to be fully elucidated. This is of important concern in broader applications of this approach, especially for neuroinflammation-sensitive studies. In this work, we first assessed the activation of microglia/macrophages for 16 weeks after microprism implantation using two-photon microscopy in awake CX3CR1-GFP mice. The imaging window became clear from bleedings after ~2 weeks and the maximum imaging distance (in the horizontal direction) stabilized at around 500 µm after ~5 weeks. We also quantified the microglial morphology from week 3 to week 16 post-implantation. Compared to non-implant controls, microglia near the microprism showed higher cell density, smaller soma, and shorter and less branched processes in the early-chronic phase. After week 5, microglial morphology further than 100 µm from the microprism was generally similar to microglia in the control group. In addition, time-lapse imaging confirmed that microglial processes were surveying normally from week 3, even for microglia as close as 50 µm away. These morphological analyses and dynamic imaging results suggest that microglia around chronically implanted microprism eventually exhibit inactive phenotypes. Next, we examined microglial/macrophage responses following laser induced micro-vessel disruptions as an example application of microprism implantation for neuroinflammation related studies. Through the microprism, we captured microglial/macrophage polarization and migration, as well as blood flow changes after the insult for additional 16 weeks. To our surprise, microglia/macrophage aggregation around the insult site was sustained over the 16-week observation period. This work demonstrates the feasibility of using microprisms for long-term characterizations of inflammatory responses to other injuries including implantable devices at deeper depths than that achievable by conventional two-photon microscopy.

Imaging the Efficiency of Poly(3,4-ethylenedioxythiophene) Doped with Acid-Functionalized Carbon Nanotube and Iridium Oxide Electrode Coatings for Microstimulation.

Electrical microstimulation has shown promise in restoring neural deficits in humans. Electrodes coated with materials like the conducting polymer poly(3,4-ethylenedioxythiophene) doped with acid-functionalized carbon nanotubes (PEDOT/CNTs, or PC) exhibit superior charge injection than traditional metals like platinum. However, the stimulation performance of PC remains to be fully characterized. Advanced imaging techniques and transgenic tools allow for real-time observations of neural activity in vivo. Herein, microelectrodes coated with PC and iridium oxide (IrOx) (a commonly used high-charge-injection material) are implanted in GCaMP6s mice and electrical stimulation is applied while imaging neuronal calcium responses. Results show that PC-coated electrodes stimulate more intense and broader GCaMP responses than IrOx. Two-photon microscopy reveals that PC-coated electrodes activate significantly more neuronal soma and neuropil than IrOx-coated electrodes in constant-voltage stimulation and significantly more neuronal soma in constant-current stimulation. Furthermore, with the same injected charge, both materials activate more spatially confined neural elements with shorter pulses than longer pulses, providing a means to tune stimulation selectivity. Finite element analyses reveal that the PC coating creates a denser and nonuniform electric field, increasing the likelihood of activating nearby neural elements. PC coating can significantly improve energy efficiency for electrical stimulation applications.