acute-brain-trauma-in-mice-followed-by-longitudinal-two-photon-imaging

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

flat-floored-air-lifted-platform-new-method-for-combining-behavior

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

a-flexible-platform-for-monitoring-cerebellum-dependent-sensory

A Flexible Platform for Monitoring Cerebellum-Dependent Sensory Associative Learning

Climbing fiber inputs to Purkinje cells provide instructive signals critical for cerebellum-dependent associative learning. Studying these signals in ...

University of Helsinki

Princeton University

Motility of mitochondria, as well as their activity-dependent immobilization ("trapping"), is essential for neuronal function, but its regulation by cytoskeleton and relevance for glial cell signalling are unknown. Using time-lapse fluorescence imaging in rat cultured astrocytes, we evaluated the role of microtubules and actin filaments in motility of mitochondria in resting cells and during physiological or pathological Ca(2+) elevations. We found that mitochondria were significantly more aligned with microtubules than with actin filaments. Mitochondria were highly mobile under resting conditions at low intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Activation of a moderate increase in [Ca(2+)](i) by either low-dose ionomycin or ATP immobilized mitochondria significantly but reversibly, without affecting mitochondrial morphology. A larger dose of ionomycin caused irreversible arrest and fragmentation of mitochondria. Disruption of microtubules completely arrested mitochondrial motility, while disruption of actin filaments had no effect on the basal mitochondrial motility at resting [Ca(2+)](i) levels but significantly reduced mitochondrial immobilization during [Ca(2+)](i) elevations. These results suggest that: (i) motility of astrocytic mitochondria is inversely related to [Ca(2+)](i), (ii) mitochondria require intact microtubules for their motility, and (iii) elevated [Ca(2+)](i) immobilizes mitochondria by strengthening their interaction with actin filaments.

Motility of astrocytic mitochondria is arrested by Ca2+-dependent interaction between mitochondria and actin filaments.

Mitochondrial DNA (mtDNA) mutations manifest with vast clinical heterogeneity. The molecular basis of this variability is mostly unknown because the lack of model systems has hampered mechanistic studies. We generated induced pluripotent stem cells from patients carrying the most common human disease mutation in mtDNA, m.3243A>G, underlying mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. During reprogramming, heteroplasmic mtDNA showed bimodal segregation toward homoplasmy, with concomitant changes in mtDNA organization, mimicking mtDNA bottleneck during epiblast specification. Induced pluripotent stem cell-derived neurons and various tissues derived from teratomas manifested cell-type specific respiratory chain (RC) deficiency patterns. Similar to MELAS patient tissues, complex I defect predominated. Upon neuronal differentiation, complex I specifically was sequestered in perinuclear PTEN-induced putative kinase 1 (PINK1) and Parkin-positive autophagosomes, suggesting active degradation through mitophagy. Other RC enzymes showed normal mitochondrial network distribution. Our data show that cellular context actively modifies RC deficiency manifestation in MELAS and that autophagy is a significant component of neuronal MELAS pathogenesis.

Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model.

Vascular changes underlying headache in migraine patients induced by Glyceryl trinitrate (GTN) were previously studied with various imaging techniques. Despite the long history of medical and experimental use of GTN, its effects on the brain vasculature are still poorly understood presumably due to low spatial resolution of the imaging modalities used so far. We took advantage of the micrometer-scale vertical resolution of two-photon microscopy to differentiate between the vasodynamic effects of GTN on meningeal versus cortical vessels imaged simultaneously in anesthetized rats through either thinned skull or glass-sealed cranial window. Intermediate and small calibre vessels were visualized in vivo by imaging intravascular fluorescent dextran, and detection of blood flow direction allowed identification of individual arterioles and venules. We found that i.p.-injected GTN induced a transient constriction of meningeal arterioles, while their cortical counterparts were, in contrast, dilated. These opposing effects of GTN were restricted to arterioles, whereas the effects on venules were insignificant. Interestingly, the NO synthase inhibitor L-NAME did not affect the diameter of meningeal vessels but induced a constriction of cortical vessels. The different cellular environment in cortex versus meninges as well as distinct vessel wall anatomical features probably play crucial role in the observed phenomena. These findings highlight differential region- and vessel-type-specific effects of GTN on cranial vessels, and may implicate new vascular mechanisms of NO-mediated primary headaches.

Opposite Reactivity of Meningeal versus Cortical Microvessels to the Nitric Oxide Donor Glyceryl Trinitrate Evaluated In Vivo with Two-Photon Imaging.

The need for automated and efficient systems for tracking full animal pose has increased with the complexity of behavioral data and analyses. Here we introduce LEAP (LEAP estimates animal pose), a deep-learning-based method for predicting the positions of animal body parts. This framework consists of a graphical interface for labeling of body parts and training the network. LEAP offers fast prediction on new data, and training with as few as 100 frames results in 95% of peak performance. We validated LEAP using videos of freely behaving fruit flies and tracked 32 distinct points to describe the pose of the head, body, wings and legs, with an error rate of <3% of body length. We recapitulated reported findings on insect gait dynamics and demonstrated LEAP's applicability for unsupervised behavioral classification. Finally, we extended the method to more challenging imaging situations and videos of freely moving mice.

Fast animal pose estimation using deep neural networks.

Spike doublets comprise ∼10% of in vivo complex spike events under spontaneous conditions and ∼20% (up to 50%) under evoked conditions. Under near-physiological slice conditions, single complex spikes do not induce parallel fibre long-term depression. Doublet stimulation is required to induce long-term depression with an optimal parallel-fibre to first-complex-spike timing interval of 150 ms.

Complex spike clusters and false-positive rejection in a cerebellar supervised learning rule.

To select actions based on sensory evidence, animals must create and manipulate representations of stimulus information in memory. Here we report that during accumulation of somatosensory evidence, optogenetic manipulation of cerebellar Purkinje cells reduces the accuracy of subsequent memory-guided decisions and causes mice to downweight prior information. Behavioral deficits are consistent with the addition of noise and leak to the evidence accumulation process. We conclude that the cerebellum can influence the accurate maintenance of working memory.

Cerebellar disruption impairs working memory during evidence accumulation.

Cerebellar outputs take polysynaptic routes to reach the rest of the brain, impeding conventional tracing. Here, we quantify pathways between the cerebellum and forebrain by using transsynaptic tracing viruses and a whole-brain analysis pipeline. With retrograde tracing, we find that most descending paths originate from the somatomotor cortex. Anterograde tracing of ascending paths encompasses most thalamic nuclei, especially ventral posteromedial, lateral posterior, mediodorsal, and reticular nuclei. In the neocortex, sensorimotor regions contain the most labeled neurons, but we find higher densities in associative areas, including orbital, anterior cingulate, prelimbic, and infralimbic cortex. Patterns of ascending expression correlate with c-Fos expression after optogenetic inhibition of Purkinje cells. Our results reveal homologous networks linking single areas of the cerebellar cortex to diverse forebrain targets. We conclude that shared areas of the cerebellum are positioned to provide sensory-motor information to regions implicated in both movement and nonmotor function.

ArticleTotal : 3	Year
Publication title Cited by 11	2014
Publication title Cited by 63	2014
Publication title Cited by 5	2022

Article	Year
Motility of astrocytic mitochondria is arrested by Ca2+-dependent interaction between mitochondria and actin filaments. Cell calcium\| PubMed ID: 23177663	2013
Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24003133	2013
Opposite Reactivity of Meningeal versus Cortical Microvessels to the Nitric Oxide Donor Glyceryl Trinitrate Evaluated In Vivo with Two-Photon Imaging. PloS one\| PubMed ID: 24586970	2014
Fast animal pose estimation using deep neural networks. Nature methods\| PubMed ID: 30573820	2019
Complex spike clusters and false-positive rejection in a cerebellar supervised learning rule. The Journal of physiology\| PubMed ID: 31297821	2019
Cerebellar disruption impairs working memory during evidence accumulation. Nature communications\| PubMed ID: 31311934	2019
Homologous organization of cerebellar pathways to sensory, motor, and associative forebrain. Cell reports\| PubMed ID: 34551311	2021

Mikhail Kislin

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