constant-voltage stimulator ds2a  (Digitimer North America LLC)

Journal: bioRxiv

Article Title: Glutamine Transport via Neurotransmitter Transporter 4 (NTT4, SLC6A17) Maintains Presynaptic Glutamate Supply at Excitatory Synapses in the Central Nervous System

doi: 10.1101/2024.10.20.616835

Figure Lengend Snippet: (A) Glutamate release from mossy fibre boutons elicits NMDA-mediated EPSCs in CA3 pyramidal cells when low-frequency (0.1 Hz; LFS; Top) and high frequency (2 Hz; HFS; Bottom) stimulation is applied to presynaptic axons. (B) Addition of leucine (10 mM; orange) to the ACSF during LFS does not lead to a reduction in mean EPSC amplitude, suggesting low frequency activity can continue independent of NTT4 over this timeframe. (C) Plot showing mean EPSC amplitudes during control (black) and following wash-in of 10 mM leucine (orange). There is no difference in EPSC amplitude during the control period (black) and after addition of 10 mM leucine (orange) when LFS is applied to presynaptic axons. (D) HFS does not affect EPSC amplitudes in slices from WT animals (black). In slices treated with leucine (orange) and slices from NTT4 KO animals (green) the EPSC amplitudes reduce, suggesting a depletion of presynaptic glutamate. (E) Plot showing mean EPSC amplitudes during LFS and during the last 5 minutes of HFS. Initial EPSC amplitudes in LFS are comparable in the different groups (Figure S5), By the end of HFS, EPSC amplitudes are significantly smaller in WT brain slices treated with leucine (orange) and in KO slices (green) whereas they have not reduced in WT control (black). The addition of leucine to KO slices (blue) causes a reduction of the same magnitude, ruling out the possibility that leucine is affecting the reduction via off-target mechanisms. The addition of 20 mM MeAIB to WT slices (yellow) caused a similar reduction of EPSC amplitude during HFS. Data presented as means ± SEM. Data points in C & E indicate individual mice. ‘ns’ = no significant difference, ‘***’ = p < 0.001 ‘****’ = p < 0.0001.

Article Snippet: EPSCs were elicited by constant-current stimulation of presynaptic axons (Digitimer DS2A stimulator) via a concentric bipolar tungsten electrode placed into the mossy fibre pathway.

Techniques: Activity Assay, Control

Journal: bioRxiv

Article Title: Glutamine Transport via Neurotransmitter Transporter 4 (NTT4, SLC6A17) Maintains Presynaptic Glutamate Supply at Excitatory Synapses in the Central Nervous System

doi: 10.1101/2024.10.20.616835

Figure Lengend Snippet: (A) Glutamate release from Schaffer collateral presynaptic terminals elicits AMPA-mediated field EPSPs (fEPSPs) in CA1 stratum radiatum when low-frequency (0.1 Hz; LFS; Top) and high frequency (1 Hz; HFS; Bottom) stimulation is applied to presynaptic axons. (B) Addition of leucine (10 mM; orange) to the ACSF during LFS does not lead to a reduction in mean fEPSP amplitude. suggesting low frequency activity can continue independent of NTT4 over this timeframe. (C) Plot showing mean fEPSP amplitudes during control (black) and following wash-in of 1 mM leucine (orange). There is no difference in fEPSP amplitude during the control period (black) and after addition of 10 mM leucine (orange) when LFS is applied to presynaptic axons. (D) HFS does not affect fEPSP amplitudes in slices from WT animals (black). In slices treated with leucine (orange) and slices from NTT4 KO animals (green), fEPSP amplitudes reduce, suggesting a depletion of presynaptic glutamate. (E) Plot showing mean fEPSP amplitudes during LFS and by the end of HFS in all groups. By the end of HFS, fEPSP amplitudes are significantly smaller in WT brain slices treated with leucine (orange) and in KO slices (green), whereas they have not reduced in WT control (black). (F) Incubation of KO slices in glutamine t-butyl ester (purple) prevents the amplitude reduction during HFS, with amplitudes remaining stable as in WT (black). (G) Mean amplitudes (mV) of fEPSPs in KO slices incubated in glutamine t-butyl ester during LFS vs the last 5 minutes of HFS show no change. All data presented as means ± SEM. Data points in C, E & G indicate individual recordings. ‘ns’ indicates no significant difference, ‘**’ = p < 0.01.

Article Snippet: EPSCs were elicited by constant-current stimulation of presynaptic axons (Digitimer DS2A stimulator) via a concentric bipolar tungsten electrode placed into the mossy fibre pathway.

Techniques: Activity Assay, Control, Incubation