Flavoprotein Autofluorescence Imaging of Neuronal Activation in the Cerebellar Cortex In Vivo

Kenneth C. Reinert; Robert L. Dunbar; Wangcai Gao; Gang Chen; Timothy J. Ebner

doi:10.1152/jn.01275.2003

Flavoprotein Autofluorescence Imaging of Neuronal Activation in the Cerebellar Cortex In Vivo

Journal of Neurophysiology ◽

10.1152/jn.01275.2003 ◽

2004 ◽

Vol 92 (1) ◽

pp. 199-211 ◽

Cited By ~ 116

Author(s):

Kenneth C. Reinert ◽

Robert L. Dunbar ◽

Wangcai Gao ◽

Gang Chen ◽

Timothy J. Ebner

Keyword(s):

Neuronal Activity ◽

Cerebellar Cortex ◽

Brain Slices ◽

Optical Signal ◽

Mitochondrial Metabolism ◽

Autofluorescence Imaging ◽

Transverse Beam ◽

Electrophysiological Response ◽

Indirect Measure

Autofluorescence has been used as an indirect measure of neuronal activity in isolated cell cultures and brain slices, but only to a limited extent in vivo. Intrinsic fluorescence signals reflect the coupling between neuronal activity and mitochondrial metabolism, and are caused by the oxidation/reduction of flavoproteins or nicotinamide adenine dinucleotide (NADH). The present study evaluated the existence and properties of these autofluorescence signals in the cerebellar cortex of the ketamine/xylazine anesthetized mouse in vivo. Surface stimulation of the unstained cerebellar cortex evoked a narrow, transverse beam of optical activity consisting of a large amplitude, short latency increase in fluorescence followed by a longer duration decrease. The optimal wavelengths for this autofluorescence signal were 420–490 nm for excitation and 515–570 nm for emission, consistent with a flavoprotein origin. The amplitude of the optical signal was linearly related to stimulation amplitude and frequency, and its duration was linearly related to the duration of stimulation. Blocking synaptic transmission demonstrated that a majority of the autofluorescence signal is attributed to activating the postsynaptic targets of the parallel fibers. Hypothesized to be the result of oxidation and subsequent reduction of flavoproteins, blocking mitochondrial respiration with sodium cyanide or inactivation of flavoproteins with diphenyleneiodonium substantially reduced the optical signal. This reduction in the autofluorescence signal was accomplished without altering the presynaptic and postsynaptic components of the electrophysiological response. Results from reflectance imaging and blocking nitric oxide synthase demonstrated that the epifluorescence signal is not the result of changes in hemoglobin oxygenation or blood flow. This flavoprotein autofluorescence signal thus provides a powerful tool to monitor neuronal activity in vivo and its relationship to mitochondrial metabolism.

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Flavoprotein autofluorescence imaging in the cerebellar cortex in vivo

Journal of Neuroscience Research ◽

10.1002/jnr.21348 ◽

2007 ◽

Vol 85 (15) ◽

pp. 3221-3232 ◽

Cited By ~ 43

Author(s):

Kenneth C. Reinert ◽

Wangcai Gao ◽

Gang Chen ◽

Timothy J. Ebner

Keyword(s):

Cerebellar Cortex ◽

Autofluorescence Imaging

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Evoked Neuronal Activity Accompanied by Transmitter Release Increases Oxygen Concentration in Rat Striatum in vivo but Not in vitro

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1992.87 ◽

1992 ◽

Vol 12 (4) ◽

pp. 629-637 ◽

Cited By ~ 22

Author(s):

Jayne B. Zimmerman ◽

Robert T. Kennedy ◽

R. Mark Wightman

Keyword(s):

Electrical Stimulation ◽

Caudate Nucleus ◽

Neuronal Activity ◽

Brain Slices ◽

Rat Striatum ◽

Initial Increase ◽

Striatal Slices ◽

Carbon Fiber Microelectrode ◽

Stimulation Of

Dopamine and oxygen (O2) were measured in the caudate nucleus of anesthetized rats and in striatal slices during electrical stimulation. Simultaneous electrochemical detection of dopamine and O2 was accomplished with fast-scan cyclic voltammetry at a Nafion-coated carbon-fiber microelectrode. Stimulation of the medial forebrain bundle resulted in synaptic overflow of dopamine in the caudate nucleus. At the same time, O2 concentration increased in the extracellular fluid with two separate phases. The amplitude of the initial increase directly correlated with the frequency of the stimulus, with the time of maximum concentration reproducible across a range of frequencies. The second increase occurred at later times with a more random amplitude and with a broad, variable shape. Agents which blocked vasodilation affected both phases: Atropine attenuated the initial increase, while the second feature was nearly absent after theophylline. Yohimbine and α-methyl- p-tyrosine did not affect the O2 responses. Local electrical stimulation of the slice preparation also resulted in dopamine overflow, but a prolonged decrease in O2 concentration accompanied this event. Striatal field stimulation in vivo produced changes in O2 concentration dependent on the relative position of the stimulating and working electrodes, but none of the responses resembled that seen in the caudate slice. Thus, while measurements in brain slices show O2 consumption as a result of stimulated neuronal activity, an apparent elevation of local cerebral blood flow during and after stimulation dominate the in vivo response.

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Rhythmic multiunit neural activity in slices of hamster suprachiasmatic nucleus reflect prior photoperiod

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.2000.278.4.r987 ◽

2000 ◽

Vol 278 (4) ◽

pp. R987-R994 ◽

Cited By ~ 54

Author(s):

Maciej Mrugala ◽

Piotr Zlomanczuk ◽

Anita Jagota ◽

William J. Schwartz

Keyword(s):

Suprachiasmatic Nucleus ◽

Neuronal Activity ◽

Brain Slices ◽

Day Length ◽

Electrophysiological Activity ◽

Discharge Rates ◽

Subjective Night ◽

The Mean

The suprachiasmatic nucleus (SCN) is an endogenous circadian pacemaker, and SCN neurons exhibit circadian rhythms of electrophysiological activity in vitro. In vivo, the functional state of the pacemaker depends on changes in day length (photoperiod), but it is not known if this property persists in SCN tissue isolated in vitro. To address this issue, we prepared brain slices from hamsters previously entrained to light-dark (LD) cycles of different photoperiods and analyzed rhythms of SCN multiunit neuronal activity using single electrodes. Rhythms in SCN slices from hamsters entrained to 8:16-, 12:12-, and 14:10-h LD cycles were characterized by peak discharge rates relatively higher during subjective day than subjective night. The mean duration of high neuronal activity was photoperiod dependent, compressed in slices from the short (8:16 and 12:12 LD) photoperiods, and decompressed (approximately doubled) in slices from the long (14:10 LD) photoperiod. In slices from all photoperiods, the mean phase of onset of high neuronal activity appeared to be anchored to subjective dawn. Our results show that the electrophysiological activity of the SCN pacemaker depends on day length, extending previous in vivo data, and demonstrate that this capacity is sustained in vitro.

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A novel dynamic network imaging analysis method reveals aging-related fragmentation of cortical networks in mouse

Network Neuroscience ◽

10.1162/netn_a_00191 ◽

2021 ◽

pp. 1-28

Author(s):

Daniel A Llano ◽

Chihua Ma ◽

Umberto Di Fabrizio ◽

Aynaz Taheri ◽

Kevin A. Stebbings ◽

...

Keyword(s):

Large Scale ◽

Brain Slices ◽

Dynamic Network ◽

Network Connectivity ◽

Analytical Tool ◽

Autofluorescence Imaging ◽

Imaging Data ◽

Cortical Networks ◽

Network Metrics

Abstract Network analysis of large-scale neuroimaging data is a particularly challenging computational problem. Here, we adapt a novel analytical tool, the community dynamic inference method (CommDy), for brain imaging data from young and aged mice. CommDy, which was inspired by social network theory, has been successfully used in other domains in biology; this report represents its first use in neuroscience. We used CommDy to investigate aging-related changes in network metrics in the auditory and motor cortices using flavoprotein autofluorescence imaging in brain slices and in vivo. We observed that auditory cortical networks in slices taken from aged brains were highly fragmented compared to networks observed in young animals. CommDy network metrics were then used to build a random-forests classifier based on NMDA-receptor blockade data, which successfully reproduced the aging findings, suggesting that the excitatory cortical connections may be altered during aging. A similar aging-related decline in network connectivity was also observed in spontaneous activity in the awake motor cortex, suggesting that the findings in the auditory cortex reflect general mechanisms during aging. These data suggest that CommDy provides a new dynamic network analytical tool to study the brain and that aging is associated with fragmentation of intracortical networks.

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Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging

PLoS Biology ◽

10.1371/journal.pbio.3000965 ◽

2020 ◽

Vol 18 (11) ◽

pp. e3000965

Author(s):

Yong Qian ◽

Danielle M. Orozco Cosio ◽

Kiryl D. Piatkevich ◽

Sarah Aufmkolk ◽

Wan-Chi Su ◽

...

Keyword(s):

Neuronal Activity ◽

In Vivo Imaging ◽

Calcium Ion ◽

Near Infrared ◽

Cultured Cells ◽

State Of The Art ◽

Brain Slices ◽

Visible Wavelength ◽

Mammalian Tissues

Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.

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Autofluorescence imaging in the mouse cerebellar cortex in vivo

Biomedical Topical Meeting ◽

10.1364/bio.2004.fc2 ◽

2004 ◽

Author(s):

T.J. Ebner ◽

W. Gao ◽

K. Reinert ◽

G. Chen

Keyword(s):

Cerebellar Cortex ◽

Autofluorescence Imaging

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Effects of L-arginine-derived nitric oxide synthesis on neuronal activity in nucleus tractus solitarius

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1995.268.2.r487 ◽

1995 ◽

Vol 268 (2) ◽

pp. R487-R491 ◽

Cited By ~ 30

Author(s):

S. Ma ◽

F. M. Abboud ◽

R. B. Felder

Keyword(s):

Nitric Oxide ◽

Neuronal Activity ◽

Nucleus Tractus Solitarius ◽

Discharge Rate ◽

Brain Slices ◽

Spontaneous Discharge ◽

Fischer 344 Rats ◽

Fischer 344

The purpose of these studies was to determine the effects of L-arginine-derived nitric oxide (NO) synthesis on neuronal activity in solitary tract nucleus (NTS) neurons. Single unit activity was recorded extracellularly from medial NTS neurons in Fischer-344 rats in vivo and in vitro. In anesthetized rats with arterial pressure maintained constant, NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg iv), an inhibitor of NO synthesis, decreased the discharge rate in 12 of 14 neurons and increased the discharge rate in two. After injection of L-NAME, the slowing of neuronal activity began within 2-5 min, and maximal responses were observed 12-15 min after injection. The decreases in activity were reversed within 12-15 min with L-arginine (30 mg/kg iv) or immediately with nitroglycerin (NTG, 10-30 micrograms/kg iv). In superfused rat brain slices, the discharge rate was reduced by 1 mM L-NAME in seven neurons, increased in two, and unchanged in one. The decreases in discharge rate were reversed by 2 mM L-arginine (4 of 6 neurons) and by 10-30 microM NTG (6 of 7 neurons). The results show that L-arginine-derived NO can affect the spontaneous discharge rate of NTS neurons. We conclude that NO may influence the excitability of NTS neurons involved in central autonomic control.

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Inversion of neurovascular coupling after subarachnoid hemorrhage in vivo

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x16686595 ◽

2017 ◽

Vol 37 (11) ◽

pp. 3625-3634 ◽

Cited By ~ 23

Author(s):

Matilde Balbi ◽

Masayo Koide ◽

George C Wellman ◽

Nikolaus Plesnila

Keyword(s):

Blood Flow ◽

Cerebral Blood Flow ◽

Subarachnoid Hemorrhage ◽

Neuronal Activity ◽

Ex Vivo ◽

Brain Slices ◽

Neurovascular Coupling ◽

Vessel Diameter ◽

Cerebral Arterioles

Subarachnoid hemorrhage (SAH) induces acute changes in the cerebral microcirculation. Recent findings ex vivo suggest neurovascular coupling (NVC), the process that increases cerebral blood flow upon neuronal activity, is also impaired after SAH. The aim of the current study was to investigate whether this occurs also in vivo. C57BL/6 mice were subjected to either sham surgery or SAH by filament perforation. Twenty-four hours later NVC was tested by forepaw stimulation and CO2 reactivity by inhalation of 10% CO2. Vessel diameter was assessed in vivo by two-photon microscopy. NVC was also investigated ex vivo using brain slices. Cerebral arterioles of sham-operated mice dilated to 130% of baseline upon CO2 inhalation or forepaw stimulation and cerebral blood flow (CBF) increased. Following SAH, however, CO2 reactivity was completely lost and the majority of cerebral arterioles showed paradoxical constriction in vivo and ex vivo resulting in a reduced CBF response. As previous results showed intact NVC 3 h after SAH, the current findings indicate that impairment of NVC after cerebral hemorrhage occurs secondarily and is progressive. Since neuronal activity-induced vasoconstriction (inverse NVC) is likely to further aggravate SAH-induced cerebral ischemia and subsequent brain damage, inverse NVC may represent a novel therapeutic target after SAH.

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