atp-sensor ateam 1.03yemk  (ATeam Scientific)

Journal: Journal of Neurochemistry

Article Title: Dynamics of Neuronal and Astrocytic Energy Molecules in Epilepsy

doi: 10.1111/jnc.70044

Figure Lengend Snippet: Neuronal cytosolic ATP levels reduced with epileptic neuronal hyperactivity. (A) An optical fiber and a pair of stimulating electrodes were implanted in the hippocampus of a transgenic mouse expressing a FRET‐based fluorescence sensor protein for cytosolic ATP in neurons (Thy1‐ATeam mouse). Screw electrodes were implanted in the skull, with their ends touching the cortical surface for EEG recordings. A train of hippocampal electrical stimulation generated epileptic neuronal hyperactivity, resulting in oscillatory neuronal discharges that persisted even after the cessation of stimulation (after‐discharges; ADs). Excitation light at ~440 nm (Blue) was delivered to the hippocampus via the optical fiber, and both CFP fluorescence (fCFP) and FRET‐mediated YFP fluorescence (fYFP) were measured. With the occurrence of epileptic neuronal hyperactivity, fYFP decreased, while fCFP increased, leading to a decrease in the calculated fYFP/fCFP ratio, suggesting a reduction in neuronal cytosolic ATP concentration. (B) Conventionally, the ratio method (fYFP/fCFP) is widely used in studies employing FRET‐based fluorescence sensor proteins. Using this method, neuronal cytosolic ATP concentration was estimated to decrease with neuronal hyperactivity. Hippocampal stimuli were delivered every hour for 12 sessions during the nighttime. The negative peak values of the ATP signal transients were measured for the first three of the 12 stimulus episodes and averaged. As described later, the series of stimulus episodes induced a kindling effect, where the latter episodes often elicited a stronger seizure response. To focus on the early stage before the kindling effect becomes prominent, only the first three episodes were examined. Data were collected from 4 animals (−0.28 ± 0.04) and are presented as mean ± SEM. (C) The YFP component of the ATeam was directly excited with ~510 nm light (Teal), allowing for measurement of the direct YFP fluorescence signal (dYFP). ATP concentration fluctuations can be estimated using either the ratio method (fYFP/fCFP) or the difference method (fYFP–dYFP). (D) Both the difference method (fYFP–dYFP) and the ratio method (fYFP/fCFP) were used to estimate the peak magnitude of neuronal cytosolic ATP reduction. Each data point represents the average of the first three stimuli of the 12‐stimulus episode series for a single animal (total of 7 animals). The diagonal line represents a linear regression fit to the data, with the XY intercept restricted at the origin (0, 0). (E) The peak times of neuronal cytosolic ATP reduction were also estimated by difference (fYFP–dYFP) and ratio (fYFP/fCFP) method. The scatter plot showed strong correlation, suggesting that either method can be reliably used to evaluate the ATP concentration transients. The diagonal line represents a linear regression fit to the data, with the XY intercept restricted at the origin (0, 0).

Article Snippet: Using fiber photometry in transgenic mice expressing the ATP sensor ATeam specifically in the cytosol of neurons (Thy1‐ATeam; Trevisiol et al. ), we observed that the CFP fluorescence (fCFP), excited by Blue light (~440 nm) increased, while the YFP fluorescence (fYFP) decreased with hippocampal train stimulation (Figure ).

Techniques: Transgenic Assay, Expressing, Fluorescence, Generated, Concentration Assay

Journal: Journal of Neurochemistry

Article Title: Dynamics of Neuronal and Astrocytic Energy Molecules in Epilepsy

doi: 10.1111/jnc.70044

Figure Lengend Snippet: ATP reduction was observed only during AD occurrences, with no correlation between the magnitude of ATP reduction and AD duration. (A) In the same animal, identical hippocampal stimulation sometimes induced AD (With AD, right) and other times did not (No AD, left). When an AD was not generated, the fluorescent signal from the ATeam showed minimal change. The inset shows that even in the ‘No AD’ case, individual electrical stimuli were effective in generating neuronal activity. When an AD was successfully generated, fluctuations in the fCFP, dYFP, and fYFP signals indicated a reduction in neuronal cytosolic ATP levels. Gray shading represents the hippocampal electrical stimulation period, while magenta shading indicates the duration of the AD. (B) The magnitude of ATP reduction with hippocampal electrical stimulation was evaluated using the difference method (fYFP‐dYFP). ATP reduction was significantly more pronounced in the ‘With AD’ condition compared to the ‘No AD’ condition recorded from the same animal (No AD: −2.43 ± 0.13, n = 6; With AD: −24.39 ± 0.38, n = 6). Similar results were obtained with another mouse; however, in this case, dYFP was not recorded. Thus, the ATP reduction was calculated with the ratio method (fYFP/fCFP) and a significant difference was observed between the conditions (No AD: −0.0068 ± 0.0026, n = 3; With AD: −0.1949 ± 0.0087, n = 9). (C) AD duration varied even with the same stimulus intensity. The cessation time of epileptic oscillatory neuronal discharges was determined through manual examination of EEG traces. The frequency band was constrained to 1–100 Hz by digital filtering and viewed at high magnification for clearer manual determination of AD cessation time, as shown in the insets. This filter effectively removed contamination from EMG signals and motion artifacts, enabling the examination of typical epileptic slow oscillations. Neuronal cytosolic ATP reduction magnitude was consistent regardless of AD duration (left: Short AD; right: Long AD). (D) The magnitude of ATP reduction was plotted against AD duration. The ATP reduction in response to the first three episodes of hippocampal stimulation in a series was examined and plotted. The red dashed line represents the calculated correlation (r = 0.32). Data from the same animal are shown in the same color, with data from 7 animals presented.

Article Snippet: Using fiber photometry in transgenic mice expressing the ATP sensor ATeam specifically in the cytosol of neurons (Thy1‐ATeam; Trevisiol et al. ), we observed that the CFP fluorescence (fCFP), excited by Blue light (~440 nm) increased, while the YFP fluorescence (fYFP) decreased with hippocampal train stimulation (Figure ).

Techniques: Generated, Activity Assay

Journal: Journal of Neurochemistry

Article Title: Dynamics of Neuronal and Astrocytic Energy Molecules in Epilepsy

doi: 10.1111/jnc.70044

Figure Lengend Snippet: Local brain blood volume (BBV) fluctuations associated with epileptic neuronal hyperactivity. (A) A train of hippocampal electrical stimulation elicited an after‐discharge (AD) response (EEG; the shown trace was band‐pass filtered at 1–100 Hz), resulting in fluctuations in fYFP (green) and dYFP (red) signals. A prominent reduction in neuronal cytosolic ATP was estimated using the difference method (fYFP–dYFP). Although dYFP from the ATeam fluorescence signal is insensitive to ATP concentration fluctuations, a notable increase in dYFP was observed during the occurrence of AD. This increase may be attributed to either local BBV reduction or neuronal cytosolic pH alkalinization. To directly evaluate BBV fluctuations, Texas Red was injected into the tail vein to visualize blood vessels (brown). As the intensity of the Texas Red fluorescence signal rapidly decayed post‐injection, a sloping baseline was fitted to adjust the Texas Red signal. The Texas Red signal decreased during the AD period, suggesting constriction of blood vessels and a subsequent reduction in local BBV adjacent to the tip of the optical fiber. (B) The timing of the positive peak of the dYFP signal (open red circle) was compared with the timing of the negative peak of the Texas Red signal (solid brown circle), both occurring at similar times. The Texas Red signal (brown trace) was inverted and represented in the middle row trace (semi‐transparent brown trace). The shape of the inverted Texas Red trace resembled that of the dYFP trace (red trace); however, it is important to note that the positive peak in the dYFP signal was much more pronounced than that in the inverted Texas Red trace. Therefore, the dYFP signal may also be influenced by pH changes in the neuronal cytosol. (C) The timing of the negative peak of the Texas Red signal was plotted against the positive peak of the dYFP signal, with a diagonal gray dashed line ( y = x ) drawn for reference. Only the first epileptic episode induced by hippocampal stimulation following the tail vein injection of Texas Red was analyzed, as the Texas Red signal was clearly detectable only during the first hour after injection. Therefore, each data point presents an individual measurement from one animal ( n = 6 mice). The positive peak in dYFP occurred either closely in time with, or preceded, the negative peak in Texas Red by as much as approximately 10 s. These data suggest that, while fluctuations in the dYFP signal primarily reflect the shadow effect from BBV changes, other factors, such as cytosolic pH, may also play a role.

Article Snippet: Using fiber photometry in transgenic mice expressing the ATP sensor ATeam specifically in the cytosol of neurons (Thy1‐ATeam; Trevisiol et al. ), we observed that the CFP fluorescence (fCFP), excited by Blue light (~440 nm) increased, while the YFP fluorescence (fYFP) decreased with hippocampal train stimulation (Figure ).

Techniques: Fluorescence, Concentration Assay, Injection

Journal: Journal of Neurochemistry

Article Title: Dynamics of Neuronal and Astrocytic Energy Molecules in Epilepsy

doi: 10.1111/jnc.70044

Figure Lengend Snippet: Rapid increase in pyruvate signal in astrocytes. (A) A FRET‐based cytosolic pyruvate fluorescence sensor was specifically expressed in astrocytes (Mlc1‐tTA::TetO‐PYRS transgenic mice). An optical fiber and a pair of stimulating electrodes were implanted into the hippocampus. Following a train of hippocampal stimulation, epileptic AD was generated (EEG; the shown trace was band‐pass filtered at 1–100 Hz), and fluctuations in the fYFP (green), fCFP (blue), and dYFP (red) signals were observed. In contrast to the binding of ATP to ATeam, FRET efficiency is expected to decrease upon the binding of pyruvate to PYRS. Astrocytic pyruvate concentration transients were estimated using either the ratio method (fCFP/fYFP) or the difference method (dYFP–fYFP). Tri‐phasic pyruvate concentration dynamics was estimated using the difference method. The onset of the first phase of the pyruvate signal increase was defined as the point where the signal exceeds the baseline. The positive peak of the pyruvate signal was also identified. (B) The positive peak of the pyruvate signal estimated using the ratio method was plotted against that obtained with the difference method ( n = 9 episodes from 3 animals). A linear regression line was fitted to the data plot with a fixed y ‐intercept at (0, 0). (C) A representative recording from a Thy1‐ATeam mouse. (D) Representative recordings of the astrocytic PYRS signal and the neuronal ATeam signal were vertically aligned on a close‐up time scale. The astrocytic pyruvate signal exhibited a transient negative deflection followed by a steep increase. The onset of the pyruvate signal increase was defined as the point where the pyruvate signal crossed the baseline. The neuronal ATP signal decreased following hippocampal stimulation. The onset of the ATP signal reduction was determined by identifying the negative peak of the second derivative of the ATP signal trace (i.e., the inflection point). Additionally, the positive peak of the pyruvate signal and the negative peak of the ATP signal were identified. (E) The onset of the first phase of the pyruvate signal increase in astrocytes (top, 2.72 ± 0.27, n = 3 animals) and the onset of the ATP signal reduction in neurons (bottom, 13.50 ± 2.83, n = 7 animals) were compared. A significant difference in the onset times was found between the astrocytic pyruvate signal and the neuronal ATP signal (Welch's t ‐test, degree of freedom = 8, t = −3.789, p = 0.008, data presented as the mean ± SEM). (F) The initial positive peak of the pyruvate signal corresponds to the onset of the signal's decrease from its maximum (top, 20.75 ± 1.31 s, n = 3 animals). This was compared with the time of the negative peak of the ATP signal in neurons (bottom, 31.86 ± 3.28 s, n = 7 animals). A significant difference was found between these times (Welch's t ‐test, degree of freedom = 8, t = −3.148, p = 0.015, data presented as the mean ± SEM). In addition to individual data points, the mean ± SEM is presented. Statistical significance was set as * p < 0.05 and ** p < 0.01.

Article Snippet: Using fiber photometry in transgenic mice expressing the ATP sensor ATeam specifically in the cytosol of neurons (Thy1‐ATeam; Trevisiol et al. ), we observed that the CFP fluorescence (fCFP), excited by Blue light (~440 nm) increased, while the YFP fluorescence (fYFP) decreased with hippocampal train stimulation (Figure ).

Techniques: Fluorescence, Transgenic Assay, Generated, Binding Assay, Concentration Assay