2-photon Laser Scanning Microscopy Research Articles

Vascular Signaling Plasticity Reprograms Blood Delivery Mechanisms to Meet Fluctuating Neuronal Energy Needs

Neuronal computation is metabolically expensive and depends on precision delivery of energy substrates—oxygen and glucose—via tightly coordinated blood flow to ensure that energy demands are continually met. This is facilitated by neurovascular coupling (NVC)—a range of mechanisms matching neural activity to local blood flow. These NVC mechanisms are typically considered invariant, and whether they are reshaped according to ever-changing neuronal metabolic needs has not been examined. We present evidence that neural activity continually reprograms the blood flow control mechanisms embedded in the local vasculature, a process we refer to as ‘vascular signaling plasticity’ (VSP). Using an established enrichment paradigm, we find that animals housed in conditions that increase input to the barrel cortex for 7 days exhibit profound neuronal plasticity in this region. This resculpts local vascular reactivity to elevate blood delivery. Indeed, VSP results in increased basal capillary blood flow and augmented activity of vasodilatory agents in vivo manifesting as increased red blood cell (RBC) flux responses to capillary stimulation and upstream diameter changes measured using 2-photon laser scanning microscopy. Moreover, the sensitivity of capillaries to stimulation is also dramatically elevated in an ex vivo isolated capillary-arteriole preparation. Using K+-mediated capillary-to-arteriole electrical signaling as a springboard to study the molecular underpinnings of VSP, we find that this process induces a ~70% increase in the density of strong inward rectifier K+ (Kir2.1) currents in capillary endothelial cells, which profoundly augments the retrograde hyperpolarization generated by these channels to control upstream diameter at the level of the penetrating arteriole. Through whole-cell patch clamp electrophysiology, we characterize the mechanism of VSP induction and recapitulate this phenomenon in vitro. We validate our findings through Crispr/Cas9-mediated endothelial cell-specific knockout of the transcription factor responsible for VSP induction. Our data thus recast the vasculature as a plastic, brain-wide activity sensing network that is continually reshaped at the molecular level by local neuronal input, leading to precisely-tuned blood delivery to meet continually fluctuating neural energy needs. VSP represents a new dimension to brain plasticity that may underpin a range of vital physiological processes and is likely disrupted in numerous brain pathologies with a blood flow component. This work was supported by the American Heart Association (23POST1023086), and NIH grants 1DP2NS121347, 1R01AG066645, and 5R01NS115401. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Label-free quantification of imaging features in the extracellular matrix of left and right-sided colon cancer tissues

The molecular pathogenesis of colorectal cancer is known to differ between the right and left side of the colon. Several previous studies have focussed on the differences in clinicopathological features, proteomic and genetic biomarkers, the composition of gut microbiota, response to therapy, and the characteristics of the tumour microenvironment. However, the morphology and density of collagen in the extracellular matrix (ECM) have not been studied intensively. In this study, we employed 2-photon laser scanning microscopy (2PLSM) to visualise the intrinsic second-harmonic generation (SHG) signal emitted by collagen fibres in the heterogeneous ECM of human colon tumour tissues. Through texture analysis of the SHG signal, we quantitatively distinguished the imaging features generated by structural differences of collagen fibres in healthy colon and cancers and found marked differences. The fibres inside of tumours exhibited a loss of organisation, particularly pronounced in right-sided colon cancer (RSCC), where the chaotic regions were significantly increased. In addition, a higher collagen content was found in left-sided colon cancer (LSCC). In future, this might aid in subclassification and therapeutic decisions or even in designing new therapy regimens by taking into account the differences between collagen fibres features between colon tumours located at different sides.

Abstract WP297: Time-Dependent Effects of Drag-Reducing Polymers Administration on Permanent Middle Cerebral Artery Occlusion Injury in Rats

Introduction: Current treatments for ischemic stroke are not focused directly on cerebral microcirculation. We previously showed that 2 μg/ml of drag-reducing polymers (DRPs) restore cerebral microcirculation and oxygenation in rats subjected to permanent middle cerebral artery occlusion (pMCAO). Here we evaluated the dependence of DRPs efficiency on stroke outcome from the time of application. Methods: DRPs (2 μg/ml) or saline was i.v. injected 0.5, 3, and 6 hrs after filament pMCAO in rats (n=10/group). In-vivo 2-photon laser scanning microscopy (2PLSM) was used to assess the acute effects of DRPs on cerebral microcirculation, hypoxia (NADH), and blood-brain barrier (BBB). Cerebral infarction, blood flow (CBF), and neurological outcomes were evaluated by magnetic resonance imaging (MRI) and behavioral testing, respectively, at 24 hours, 1 and 3 weeks after pMCAO. Differences between groups were determined using a two-way analysis of variance for multiple comparisons and posthoc testing with the statistical significance level set at p<0.05. Results: In vivo 2PLSM showed that pMCAO progressively decreased cerebral microcirculation leading to hypoxia and BBB disruption (p<0.05 from baseline). DRPs (0.5-6 hrs) increased near-wall blood flow velocity and flow rate in arterioles, leading to an increase in the number of erythrocytes entering capillaries, capillary perfusion and oxygenation while protecting BBB compared to saline (p<0.05). MRI demonstrated reduced infarct expansion (T2) and mitigated CBF disturbances (arterial spin labeling) in all DRPs-treated groups compared to saline (p<0.05). Neurobehavioral testing revealed more preserved neurological function in all DRPs groups than in the saline group (p<0.05). The obtained results showed that DRPs efficacy reduces with the time of application from 0.5 h to 6 hours after pMCAO but remains significant (p<0.05). Conclusions: DRPs (effectively restore cerebral microcirculation after pMCAO reducing tissue hypoxia and BBB damage, leading to reduced infarct size and improved neurological outcome in a time-dependent manner. DRPs can be used as a new effective adjunct therapy for ischemic stroke even without reperfusion and after delayed administration following the stroke onset.

EXTH-25. IN VIVO TWO-PHOTON TUMOR IMAGING IN AN ORTHOTOPIC MEDULLOBLASTOMA MOUSE MODEL

Abstract BACKGROUND Medulloblastoma represents the most common primary brain malignancy in pediatric patients and is associated with neuro-cognitive impairment and frequent relapses after aggressive multimodal therapy. Adoptive immunotherapies such as CAR T-cell therapy are currently being investigated, showing promising results in preclinical models but lacking efficacy in first-in-human trials. Refined in vivo models to investigate reasons for treatment failures are therefore urgently needed. METHODS We developed a xenogeneic orthotopic medulloblastoma (MB) model in mice by combining a chronic cerebellar window with repetitive intravital two-photon laser scanning microscopy. Red tdTomato-fluorescent DAOY (SHH MB) tumor cells expressing B7-H3 were implanted intracranially into the cerebellum of immunodeficient mice (n = 7), and tumor cell formation was followed by in vivo-microscopy. Intravenous fluorescein isothiocyanate (FITC)-dextran was used for intravascular plasma staining. B7-H3-directed CAR T-cells were injected into the adjacent brain parenchyma. RESULTS Chronic cranial window implantation was well tolerated and allowed repetitive visualization of the mouse cerebellum. After intracranial tumor cell implantation, continuous tumor growth could be evaluated with epifluorescence as well as 2-photon laser scanning microscopy. Tumor formation was identified as early as day 5 in every mouse and could continuously be followed up to 35 days until mice succumbed due to tumor burden. Window quality and fluorescence intensity remained high until the end of the experiments. Studies on GFP-expressing CAR T-cell injection directed against B7-H3 are currently ongoing. CONCLUSIONS We herein establish a robust orthotopic medulloblastoma mouse model that allows repetitive in vivo tracking of fluorescence-expressing tumor cells, CAR T-cells and blood vessels over weeks. Such models may be used to study medulloblastoma tumor growth under the influence of different immunotherapies and reveal interactions between tumor cells and CAR T-cell therapies on a single-cell level.

Chronic Administration of Ion Channel Blockers Impact Microglia Morphology and Function in a Murine Model of Alzheimer's Disease.

As the population ages, a high prevalence of multimorbidity will affect the way physicians need to think about drug interactions. With microglia's important involvement in the pathology and progression of Alzheimer's disease (AD), understanding whether systemically administered drugs intended for other affections could impact microglia function, already impacted by the presence of beta-amyloid, is important. The aim of this study was to evaluate morphological changes of microglia, using in vivo 2-photon laser scanning microscopy, in a murine model of AD under systemic administration of sodium or calcium ion channel blockers in order to establish potential effects that these drugs might have on microglia under neuro-inflammatory conditions. A total of 30 mice (age 14-16 weeks, weight 20-25 g) were used, with 25 APP randomly divided into three groups. The remaining animals were CX3CR1GFP/GFP male mice (n = 5) used as WT controls. After baseline behavior testing, all animals received daily intraperitoneal injections for 30 days according to the assigned group [WT (n = 5), Control (n = 5), Carbamazepine (n = 10), and Verapamil (n = 10)]. The results showed that the Verapamil treatment improved short-term memory and enhanced exploratory behavior in APP mice. The Carbamazepine treatment also improved short-term memory but did not elicit significant changes in anxiety-related behavior. Both Verapamil and Carbamazepine reduced the surveillance speed of microglia processes and changed microglia morphology in the cortex compared to the Control group. Due to their complex molecular machinery, microglia are potentially affected by drugs that do not target them specifically, and, as such, investigating these interactions could prove beneficial in our management of neurodegenerative pathologies.

OS09.1.AIN VIVOTWO-PHOTON TUMOR IMAGING IN AN ORTHOTOPIC MEDULLOBLASTOMA MOUSE MODEL

Abstract BACKGROUND Medulloblastoma represents the most common primary brain malignancy in pediatric patients and is associated with neuro-cognitive impairment and frequent relapses after aggressive multimodal therapy. Adoptive immunotherapies such as CAR T-cell therapy are currently being investigated, showing promising results in preclinical models but lacking efficacy in first-in-human trials. Refined in vivo models to investigate reasons for treatment failures are therefore urgently needed. METHODS We developed a xenogeneic orthotopic medulloblastoma (MB) model in mice by combining a chronic cerebellar window with repetitive intravital two-photon laser scanning microscopy. Red tdTomato-fluorescent DAOY (SHH MB) tumor cells expressing EphA2 were implanted intracranially into the cerebellum of immunodeficient mice (n = 7), and tumor cell formation was followed by in vivo-microscopy. Intravenous fluorescein isothiocyanate (FITC)-dextran was used for intravascular plasma staining. EphA-directed CAR T-cells were injected into the adjacent brain parenchyma. RESULTS Chronic cranial window implantation was well tolerated and allowed repetitive visualization of the mouse cerebellum. After intracranial tumor cell implantation, continuous tumor growth could be evaluated with epifluorescence as well as 2-photon laser scanning microscopy. Tumor formation was identified as early as day 5 in every mouse and could continuously be followed up to 35 days until mice succumbed due to tumor burden. Window quality and fluorescence intensity remained high until the end of the experiments. Studies on GFP-expressing CAR T-cell injection directed against EphA2 are currently ongoing. CONCLUSIONS We herein establish a robust orthotopic medulloblastoma mouse model that allows repetitive in vivo tracking of fluorescence-expressing tumor cells, CAR T-cells and blood vessels over weeks. Such models may be used to study medulloblastoma tumor growth under the influence of different immunotherapies and reveal interactions between tumor cells and CAR T-cell therapies on a single-cell level.

Bimodal modulation of L1 interneuron activity in anterior cingulate cortex during fear conditioning.

The anterior cingulate cortex (ACC) plays a crucial role in encoding, consolidating and retrieving memories related to emotionally salient experiences, such as aversive and rewarding events. Various studies have highlighted its importance for fear memory processing, but its circuit mechanisms are still poorly understood. Cortical layer 1 (L1) of the ACC might be a particularly important site of signal integration, since it is a major entry point for long-range inputs, which is tightly controlled by local inhibition. Many L1 interneurons express the ionotropic serotonin receptor 3a (5HT3aR), which has been implicated in post-traumatic stress disorder and in models of anxiety. Hence, unraveling the response dynamics of L1 interneurons and subtypes thereof during fear memory processing may provide important insights into the microcircuit organization regulating this process. Here, using 2-photon laser scanning microscopy of genetically encoded calcium indicators through microprisms in awake mice, we longitudinally monitored over days the activity of L1 interneurons in the ACC in a tone-cued fear conditioning paradigm. We observed that tones elicited responses in a substantial fraction of the imaged neurons, which were significantly modulated in a bidirectional manner after the tone was associated to an aversive stimulus. A subpopulation of these neurons, the neurogliaform cells (NGCs), displayed a net increase in tone-evoked responses following fear conditioning. Together, these results suggest that different subpopulations of L1 interneurons may exert distinct functions in the ACC circuitry regulating fear learning and memory.

LPS-Induced Systemic Inflammation Affects the Dynamic Interactions of Astrocytes and Microglia with the Vasculature of the Mouse Brain Cortex.

The Neurovascular Unit (NVU), composed of glia (astrocytes, oligodendrocytes, microglia), neurons, pericytes and endothelial cells, is a dynamic interface ensuring the physiological functioning of the central nervous system (CNS), which gets affected and contributes to the pathology of several neurodegenerative diseases. Neuroinflammation is a common feature of neurodegenerative diseases and is primarily related to the activation state of perivascular microglia and astrocytes, which constitute two of its major cellular components. Our studies focus on monitoring in real time the morphological changes of perivascular astrocytes and microglia, as well as their dynamic interactions with the brain vasculature, under physiological conditions and following systemic neuroinflammation triggering both microgliosis and astrogliosis. To this end, we performed 2-photon laser scanning microscopy (2P-LSM) for intravital imaging of the cortex of transgenic mice visualizing the dynamics of microglia and astroglia following neuroinflammation induced by systemic administration of the endotoxin lipopolysaccharide (LPS). Our results indicate that following neuroinflammation the endfeet of activated perivascular astrocytes lose their close proximity and physiological cross-talk with vasculature, an event that most possibly contributes to a loss of blood-brain barrier (BBB) integrity. At the same time, microglial cells become activated and exhibit a higher extent of physical contact with the blood vessels. These dynamic responses of perivascular astrocytes and microglia are peaking at 4 days following LPS administration; however, they still persist at a lower level at 8 days after LPS injection, revealing incomplete reversal of inflammation affecting the glial properties and interactions within the NVU.

Transient astrocytic accumulation of fluorescein during spreading depolarizations

Spreading depolarizations (SDs) occur frequently in acute cerebral injuries. They are characterized by a breakdown of transmembrane ion gradients resulting in a reduced extracellular sodium ([Na+]o) and increased extracellular potassium concentration ([K+]o). Elevated [K+]o induces astrocytic swelling, another feature of SD; however, the solutes that drive astrocytic swelling remain incompletely understood. We incidentally found astrocytic accumulation of fluorescein (Fluo) – a low molecular weight anionic dye – during SDs induced by elevated [K+]o. Herein, we aimed to explore the properties of astrocytic Fluo accumulation during SDs, electrical stimulation, [K+]o and glutamate elevation and elucidate underlying mechanisms and its relation to swelling. Experiments were performed in acute neocortical slices from adult male C57Bl6 mice and transgenic mice expressing tdTomato in parvalbumin (PV)-positive neurons. We labeled astrocytes with sulforhodamine-101 (SR-101), measured Fluo kinetics using 2-photon laser scanning microscopy and recorded local field potentials (LFP) to detect SDs. Elevations of [K+]o lead to an increase of the astrocytic Fluo intensity in parallel with astrocytic swelling. Pharmacological inhibitors of sodium‑potassium ATPase (Na/K-ATPase), secondary-active transporters and channels were used to address the underlying mechanisms. Fluo accumulation as well as swelling were only prevented by inhibition of the sodium‑potassium ATPase. Application of glutamate or hypoosmolar solution induced astrocytic swelling independent of Fluo accumulation and glutamate opposed Fluo accumulation when co-administered with high [K+]o. Astrocytes accumulated Fluo and swelled during electrical stimulation and even more during SDs. Taken together, Fluo imaging can be used as a tool to visualize yet unidentified anion fluxes during [K+]o- but not glutamate- or hypoosmolarity induced astrocytic swelling. Fluo imaging may thereby help to elucidate mechanisms of astrocytic swelling and associated fluid movements between brain compartments during physiological and pathological conditions, e.g. SDs.

1228: MITIGATION OF POST-SEPTIC MICROCIRCULATION DYSFUNCTION BY DRAG-REDUCING POLYMERS

Introduction: Sepsis and septic shock in multiorgan dysfunction syndrome (MODS) are characterized by inflammation, coagulopathy, and vascular collapse with the endothelial and microvascular breakdown of endothelial function, the primary cause of mortality among hospitalized patients. We evaluated here whether drag-reducing polymers (DRP) can alleviate sepsis and MODS-associated panvascular dysregulation of microvascular blood flow using a mouse model of LPS-induced sepsis. Since the pathophysiology of MODS and Covid-19 share many characteristics in inflammation, coagulopathy, and low blood flow as a constellation of factors culminating in low microvascular shear rate and loss of endothelial function common to the pathophysiology of both diseases, the model utilized in this study also can be considered as the Covid-19 model. Methods: To induce acute sepsis and MODS, lipopolysaccharide (LPS-Salmonella Thyphosa) was injected i.v. (10 mg/kg) in C57BL/6J mice. In-vivo 2-photon laser scanning microscopy was used to monitor systemic cerebral (parietal cortex) and peripheral (ear) microcirculation, NADH (hypoxia), and oxidative stress. Blood samples obtained at autopsy were analyzed for Inflammation, coagulopathy, and endothelial glycocalyx disintegration biomarkers. Brain, lungs, kidney, liver, muscle, and intestine (rectum) were histologically evaluated. Differences between groups were determined using two-way ANOVA for multiple comparisons and post-hoc testing using the Mann-Whitney U-test. Results: LPS injection induced inflammatory reaction and microvascular dysfunction. DRP alleviated the inflammation, microthrombosis formation, and microvascular dysfunction in all organs evaluated (p< 0.05). Blood samples analysis by ELISA revealed reduced inflammation, coagulopathy, and endothelial glycocalyx disintegration in the DRP-treated group (p< 0.05). Conclusions: Hemorheological modulation of blood flow by DRP effectively improves systemic and peripheral circulation, reducing microthrombosis formation, inflammation and microvascular dysfunction, alleviating sepsis and MODS.

Functional α7 nicotinic acetylcholine receptors in GABAergic neurons of the interpeduncular nucleus

The interpeduncular nucleus (IPN) plays a key role in nicotine dependence and is involved in regulation of fear responses, affective states, and novelty processing. IPN neurons express nicotinic acetylcholine receptors (nAChR) and receive strong cholinergic innervation from the ventral medial habenula. Dorsal medial habenula neurons are primarily peptidergic, releasing substance P (SP) mainly onto IPN neurons in the lateral subnucleus (IPL). IPL neurons are sensitive to SP, but it is not known if they are involved in cholinergic transmission like other IPN neurons. We examined nAChR subunit gene expression in IPL neurons, revealing that Chrna7 (α7 nAChR subunit) is expressed in a subset of GABAergic IPL neurons. In patch-clamp recordings from IPL neurons, ACh-evoked inward currents were attenuated by methyllycaconitine (α7 nAChR antagonist) and potentiated by NS1738 (α7 Type I positive allosteric modulator). We confirmed α7 functional expression in IPL neurons by also showing that ACh-evoked currents were potentiated by PNU-120596 (Type II positive allosteric modulator). Additional pharmacological experiments show that IPN neurons expressing α7 nAChRs also express α3β4 nAChRs. Finally, we used 2-photon laser scanning microscopy and nicotine uncaging to directly examine the morphology of IPL neurons that express α7 nAChRs. These results highlight a novel aspect of α7 nAChR neurobiology, adding to the complexity of cholinergic modulation by nAChRs in the IPN.

Haemorheologic Enhancement of Cerebral Perfusion Improves Oxygen Supply and Reduces Aβ Plaques Deposition in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a consequence of complex interactions of age-related neurodegeneration and vascular-associated pathologies, affecting more than 44 million people worldwide. For the last decade, it has been suggested that chronic brain hypoperfusion and consequent hypoxia play a direct role in the pathogenesis of AD. However, current treatments of AD have not focused on restoring or improving microvascular perfusion. In a previous study, we showed that drag reducing polymers (DRP) enhance cerebral blood flow and tissue oxygenation. We hypothesised that haemorheologic enhancement of cerebral perfusion by DRP would be useful for treating Alzheimer's disease. We used double transgenic B6C3-Tg(APPswe, PSEN1dE9) 85Dbo/Mmjax AD mice. DRP or vehicle (saline) was i.v. injected every week starting at four months of age till 12months of age (10 mice/group). In-vivo 2-photon laser scanning microscopy was used to evaluate amyloid plaques development, cerebral microcirculation, and tissue oxygen supply/metabolic status (NADH autofluorescence). The imaging sessions were repeated once a month till 12months of age. Statistical analyses were done by independent Student's t-test or Kolmogorov-Smirnov tests where appropriate. Differences between groups and time were determined using a two-way repeated measures ANOVA analysis for multiple comparisons and post hoc testing using the Mann-Whitney U test. In the vehicle group, numerous plaques completely formed in the cortex by nine months of age. The development of plaques accumulation was accompanied by cerebral microcirculation disturbances, reduction in tissue oxygen supply and metabolic impairment (NADH increase). DRP mitigated microcirculation and tissue oxygen supply reduction - microvascular perfusion was 29.5±5%, and tissue oxygen supply was 22±4% higher than in the vehicle group (p<0.05). In the DRP group, amyloid plaques deposition was substantially less than in the vehicle group (p<0.05). Thus, rheological enhancement of blood flow by DRP is associated with reduced rate of beta amyloid plaques deposition in AD mice.

Complete Visualization of T Follicular Helper Cells in Germinal Centers by Light Sheet Fluorescence Microscopy.

Long-lasting immunity depends on generation of antibody forming cells in germinal centers (GCs). Conventional methods such as immunohistology and intravital live imaging have been used extensively to investigate the location of cellular assemblies within tissues as well as their dynamic motility and cellular interactions. Two photon laser scanning microscopy (TPLSM) intravital imaging allows scanning of large areas within tissues and reveals multiple immune cell niches. Nonetheless, this type of imaging is limited by the depth of penetration and cannot capture effectively all of the GC niches within lymphoid organs. Here we describe a method to visualize antigen-specific T and B cells in multiple microanatomical locations and niches at the level of a whole organ. This large-scale imaging approach can greatly increase our understanding of the spatial distribution of immune cells and help obtain detailed 3D maps of their locations and quantities.

In vivo two-photon characterization of tumor-associated macrophages and microglia (TAM/M) and CX3CR1 during different steps of brain metastasis formation from lung cancer.

Brain metastases frequently occur in lung cancer and dramatically limit prognosis of affected patients. The influence of tumor-associated macrophages and microglia (TAM/M) and their receptor CX3CR1 on different steps of brain metastasis formation from lung cancer is poorly characterized. We established a syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intravital 2-photon laser scanning microscopy. This allowed in vivo tracking of fluorescence-expressing tumor cells and TAM/M on a single-cell level over weeks. Intracarotid injection of red tdTomato-fluorescent Lewis lung carcinoma cell was performed in transgenic mice either proficient or deficient for CX3CR1. After intracarotid cell injection, intravascular tumor cells extravasated into the brain parenchyma and formed micro- and mature macrometastases. We observed potential phagocytosis of extravasated tumor cells by TAM/M. However, during later steps of metastasis formation, these anti-tumor effects diminished and were paralleled by TAM/M accumulation and activation. Although CX3CR1 deficiency resulted in a lower number of extravasated tumor cells, progression of these extravasated cells into micro metastases was more efficient. Overall, this resulted in a comparable number of mature macrometastases in CX3CR1-deficient and -proficient mice. Our findings indicate that unspecific inhibition of CX3CR1 might not be a suitable therapeutic option to prevent dissemination of lung cancer cells to the brain. Given the close interaction between TAM/M and tumor cells during metastasis formation, other therapeutic approaches targeting TAM/M function may warrant further evaluation. The herein established orthotopic mouse model may be a useful tool to evaluate such concepts in vivo.

P10.02 Combined methods of a micropump system and a chronic cranial window allows tumor observation with multi photon laser scanning microscopy under continuous treatment

Abstract BACKGROUND Glioblastomas are notoriously therapy resistant tumors. As opposed to other tumor entities, no major advances in therapeutic success have been made in the past decades. This has been calling for a deeper biological understanding of the tumor, its growth and resistance patterns. We have been using a xenograft glioma model, where human glioblastoma cells are implanted under chronic cranial windows and studied longitudinally over many weeks and months using multi photon laser scanning microscopy (MPLSM). To test the effect of (new) drugs, a stable and direct delivery system avoiding the blood-brain-barrier has come into our interest. MATERIAL AND METHODS We implanted cranial windows and fluorescently labeled human glioblastoma stem-like cells into NMRI nude mice to follow up on the tumor development in our MPLSM model. After tumor establishment, an Alzet® micropump was implanted to directly deliver agents via a catheter system continuously over 28 days directly under the cranial window onto the brain surface. Using the MPLSM technique, the continuous delivery and infusion of drugs onto the brain and into the tumor was measured over many weeks in detail using MPLSM. RESULTS The establishment of the combined methods allowed reliable concurrent drug delivery over 28 days bypassing the blood-brain-barrier. Individual regions and tumor cells could be measured and followed up before, and after the beginning of the treatment, as well as after the end of the pump activity. Fluorescently labelled drugs were detectable in the MPLSM and its distribution into the brain parenchyma could be quantified. After the end of the micropump activity, further MPLSM measurements offer the possibility to observe long term effects of the applied drug on the tumor. CONCLUSION The combination of tumor observation in the MPSLM and concurrent continuous drug delivery is a feasible and reliable method for the investigation of (novel) anti-tumor agents, especially drugs that are not blood-brain-barrier penetrant. Morphological or even functional changes of individual tumor cells can be measured under and after treatment. These techniques can be used to test new drugs targeting the tumor, its tumor microtubes and tumor cells networks, and measure the effects longitudinally.

Changes in Synaptic Terminal Structure in Adolescent Rat during Pregnancy; The Action Potential Propagation and Synaptic Transmission

Synaptic plasticity is a biological system of specific pattern of synaptic activity result in changes in synaptic strength. This influence puberty, pregnancy hormones, sensory experiences, and brain disorders. Long-term synaptic plasticity is accompanied by protein synthesis and trafficking, leading to structural changes of the synapse. Increasing evidence connects the terminal synaptic changes with potential propagation in adolescent and pregnancy. We investigate on the synaptic structural plasticity, which has mainly been studied with in vivo two photon laser scanning microscopy. We also discuss how a different type of synapses, the multicontact synapses associated with pregnancy.

Feed-forward recruitment of electrical synapses enhances synchronous spiking in the mouse cerebellar cortex.

In the cerebellar cortex, molecular layer interneurons use chemical and electrical synapses to form subnetworks that fine-tune the spiking output of the cerebellum. Although electrical synapses can entrain activity within neuronal assemblies, their role in feed-forward circuits is less well explored. By combining whole-cell patch-clamp and 2-photon laser scanning microscopy of basket cells (BCs), we found that classical excitatory postsynaptic currents (EPSCs) are followed by GABAA receptor-independent outward currents, reflecting the hyperpolarization component of spikelets (a synapse-evoked action potential passively propagating from electrically coupled neighbors). FF recruitment of the spikelet-mediated inhibition curtails the integration time window of concomitant excitatory postsynaptic potentials (EPSPs) and dampens their temporal integration. In contrast with GABAergic-mediated feed-forward inhibition, the depolarizing component of spikelets transiently increases the peak amplitude of EPSPs, and thus postsynaptic spiking probability. Therefore, spikelet transmission can propagate within the BC network to generate synchronous inhibition of Purkinje cells, which can entrain cerebellar output for driving temporally precise behaviors.

The Effects of Propofol on Neural Responses in the Mouse Primary Auditory Cortex.

Two-photon laser-scanning microscopy allows for the monitoring of all brain neurons with single-cell and single-action potential accuracy. This study aimed to investigate the neural responses of the primary auditory cortex to sound stimuli in awake and propofol-anesthetized mice using 2-photon laser-scanning microscopy. Twelve healthy adult male C57BL/6 mice were used in the present study. In each mouse, the scalp was removed over the entire dorsal skull, and the right primary auditory cortex (A1) located. The test stimulus, used in the awake and propofol-induced anesthetic state, was a group of tones with a random combination of 3 sound intensities and 8 sound frequencies. The calcium indicator GCaMP6s was virally expressed in cortical neurons and neuronal activity was recorded using 2-photon imaging. Calcium responses to sound stimuli in two thirds of the neuronal population of the A1 layer were significantly inhibited by propofol anesthesia. In a single neuron, the calcium responses were also inhibited by propofol anesthesia. In the waking state, △F/F (where F is the time series of fluorescence intensity) of all single neurons was significantly higher than that in the propofol-induced anesthetic state (n=669, P<0.001). Finally, in one example session and averaged across different fields of views (n=6 sessions), the response events to sound stimuli were also inhibited by propofol anesthesia. Anesthetic doses of propofol inhibited calcium transients and neuronal activity in the primary auditory cortex of mice.

Resuscitation with Drag Reducing Polymers after Traumatic Brain Injury with Hemorrhagic Shock Reduces Microthrombosis and Oxidative Stress.

Outcome after traumatic brain injury (TBI) is worsened by hemorrhagic shock (HS); however, the existing volume expansion approach with resuscitation fluids (RF) is controversial as it does not adequately alleviate impaired microvascular cerebral blood flow (mCBF). We previously reported that resuscitation fluid with drag reducing polymers (DRP-RF) improves CBF by rheological modulation of hemodynamics. Here, we evaluate the efficacy of DRP-RF, compared to lactated Ringers resuscitation fluid (LR-RF), in reducing cerebral microthrombosis and reperfusion mitochondrial oxidative stress after TBI complicated by HS. Fluid percussion TBI (1.5 ATA, 50ms) was induced in rats and followed by controlled HS to a mean arterial pressure (MAP) of 40mmHg. DRP-RF or LR-RF was infused to restore MAP to 60mmHg for 1h (pre-hospital period), followed by blood re-infusion to a MAP=70mmHg (hospital period). In vivo 2-photon laser scanning microscopy over the parietal cortex was used to monitor microvascular blood flow, nicotinamide adenine dinucleotide (NADH) for tissue oxygen supply and mitochondrial oxidative stress (superoxide by i.v. hydroethidine [HEt], 1mg/kg) for 4h after TBI/HS, followed by Dil vascular painting during perfusion-fixation. TBI/HS decreased mCBF resulting in capillary microthrombosis and tissue hypoxia. Microvascular CBF and tissue oxygenation were significantly improved in the DRP-RF compared to the LR-RF treated group (p<0.05). Reperfusion-induced oxidative stress, reflected by HEt fluorescence, was 32±6% higher in LR-RF vs. DRP-RF (p<0.05). Post-mortem whole-brain visualization of DiI painted vessels revealed multiple microthromboses in both hemispheres that were 29±3% less in DRP-RF vs. LR-RF group (p<0.05). Resuscitation after TBI/HS using DRP-RF effectively restores mCBF, reduces hypoxia, microthrombosis formation, and mitochondrial oxidative stress compared to conventional volume expansion with LR-RF.

Invivo two-photon imaging of neuronal and brain vascular responses in mice chronically exposed to ethanol.

The effects of ethanol on brain function have been extensively studied using a variety of invitro and invivo techniques. For example, electrophysiological studies using brain slices from rodents and non-human primates have demonstrated that acute and chronic exposure to ethanol alters the intrinsic excitability and synaptic signaling of neurons within cortical and sub-cortical areas of the brain. In humans, neuroimaging studies reveal alterations in measures of brain activation and connectivity in subjects with alcohol use disorder. While complementary, these methods are inherently limited due to issues related to either disruption of normal sensory input (invitro slice studies) or resolution (whole brain imaging). In the present study, we used 2-photon laser scanning microscopy in intact animals to assess the impact of chronic ethanol exposure on sensory-evoked neuronal and vascular responses. Adult male C57BL/6J mice were exposed to four weekly cycles of chronic intermittent ethanol (CIE) exposure, while control mice were exposed to air. After withdrawal (≥72h), a cranial window was placed over the primary visual cortex (V1), and sensory-evoked responses were monitored using the calcium indicator OGB-1. CIE exposure produced small but significant changes in response amplitude (decrease) and orientation selectivity of V1 neurons (increase). While arteriole diameter did not differ between control and CIE mice under baseline conditions, sensory-evoked dilation was enhanced in vessels from CIE-exposed mice as compared to controls. This was accompanied by a reduced latency in response to stimulation. In separate experiments, pial arteriole diameter was measured in the barrel cortex of control and CIE-exposed mice. Baseline diameter of barrel cortex arterioles was similar between control and CIE-exposed mice, but unlike vessels in V1, sensory-evoked dilation of barrel cortex arterioles was similar between the two groups. Together, the results of these studies suggest that chronic exposure to alcohol induces changes in neurovascular coupling that are region-dependent.