Datos de investigación
Electrophysiological recordings of Drosophila accesory medulla neurons
Publicador:
Consejo Nacional de Investigaciones Científicas y Técnicas
Fecha de depósito:
01/04/2025
Fecha de creación:
2013/2023
Clasificación temática:
Resumen
These data corresponds to raw electrophysiological recordings of accessory medulla neurons of Drosohila melanogaster under different conditions (voltage/current protocols, pharmacological treatments). Recordings were acquired throught whole-cell patch clamp following established methodology (Patch-Clamping Drosophila Brain Neurons, Fernandez-Chiappe, Muraro 2022 Aug 1;2022(8):pdb.prot107936. doi: 10.1101/pdb.prot107936).
Métodos
Patch clamp experiments were conducted as previously described. Briefly, 3-to-9 day old female flies were anesthetized with a short incubation of the vial on ice, and brain dissection was performed in external recording solution, which consisted of the following, in mM: 101 NaCl, 3 KCl, 1 CaCl2, 4 MgCl2, 1.25 NaH2PO4, 5 glucose, and 20.7 NaHCO3, pH 7.2, with an osmolarity of 250 mmol/kg. After removal of the proboscis, air sacks, and head cuticle, the brain was glued ventral side up to a Sylgard-coated coverslip using a few microliters of tissue adhesive Vetbond (3M). The time from anesthesia to the establishment of the recordings, that we term time since dissection, was spent as follows: LNvs were visualized by red fluorescence in Pdf-RFP flies, which express a red fluorophore under the Pdf promoter, using an Olympus BX51WI upright microscope with 60$\times$ water-immersion lens, and ThorLabs LEDD1B and TK-LED (TOLKET S.R.L.) illumination systems. Once the fluorescent cells were identified, cells were visualized under IR-DIC using a DMK23UP1300 Imaging Source camera and IC Capture 2.4 software. lLNvs were distinguished from sLNvs by their size and anatomic position. To allow access of the recording electrode, the superficial glia directly adjacent to LNvs somas were locally digested with protease XIV solution (10 mg/ml; P5147, Sigma-Aldrich) dissolved in external recording solution. This was achieved using a large opened tip (20 um) glass capillary (pulled from glass of the type FG-GBF150-110-7.5; Sutter Instrument) and gentle massage of the superficial glia with mouth suction to render the underlying cell bodies accessible for the recording electrode with minimum disruption of the neuronal circuits. After this procedure, the protease solution was quickly washed by perfusion of the external solution using a peristaltic pump (catalog \#ISM831, ISMATEC). Recordings were performed using thick-walled borosilicate glass pipettes (FG-GBF150-86-7.5, Sutter Instrument) pulled to 7-8 M$\Omega$ using a horizontal puller P-97 (Sutter Instrument) and fire polished to 9-12 M$\Omega$. Recordings were made using a Multiclamp 700B amplifier controlled by pClamp 10.4 software via an Axon Digidata 1515 analog-to-digital converter (Molecular Devices) and saved into .abf files. Recording pipettes were filled with internal solution containing the following (in mM): 102 potassium gluconate, 17 NaCl, 0.085 CaCl2, 0.94 EGTA, and 8.5 HEPES, pH 7.2, with an osmolarity of 235 mmol/kg. Gigaohm seals were accomplished using minimal suction followed by break-in into the whole-cell configuration using gentle suction in voltage-clamp mode with a holding voltage of -60mV. Spontaneous firing was recorded in current clamp ($I = 0$) mode. For the dual recording experiments, the sequence was done two consecutive times, achieving the whole-cell patch clamp configuration in one neuron after the other (in the case of sLNv-lLNv recordings, the lLNv was patched in the first place). Once both neurons were patched, the electrical activity was recorded for both neurons simultaneously. Simultaneous recordings were always performed in neurons located ipsilaterally. For the mecamylamine (M9020, Sigma-Aldrich) and picrotoxin (P1675, Sigma-Aldrich) experiments, after 1 to 3 min of recording basal conditions that served as a control, 10 ml of mecamylamine (10 uM) solution, or 10 ml of picrotoxin (100 uM) solution, both prepared in external saline, were perfused over 3 min. Drugs were then washed out with external saline perfusion. Perfusion was continuous throughout the experiments. For the phase response curve experiments, neurons were initially recorded for 1 to 3 minutes to determine their baseline bursting frequency (bursts per 60 seconds). The stimulation frequency was then calculated by dividing the baseline bursting frequency by 5. To ensure that the stimulation frequency did not coincide with the neuron's spontaneous firing, the value was rounded, which resulted in a shift of +- 0.01 to 0.02 Hz. This adjustment prevented the stimulation frequency from being an exact multiple of the baseline firing frequency, so that stimulation would be unlikely to fall in the same part of the cycle repeatedly. Once the stimulation frequency was determined, neuronal stimulation was performed using pulses of -20 pA amplitude and 100 ms duration. Each stimulation trial consisted of 5 sweeps, each lasting 90 seconds. After the final sweep of stimulation, it was verified that the neuron continued to fire spontaneous bursts with a frequency similar to the baseline frequency recorded at the beginning.
Alcance geográfico
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Identificador del recurso
Colecciones
Datos de Investigación(IBIOBA - MPSP)
Datos de Investigación de INST. D/INV.EN BIOMED.DE BS AS-CONICET-INST. PARTNER SOCIEDAD MAX PLANCK
Datos de Investigación de INST. D/INV.EN BIOMED.DE BS AS-CONICET-INST. PARTNER SOCIEDAD MAX PLANCK
Datos de Investigación(IFIBA)
Datos de Investigación de INST.DE FISICA DE BUENOS AIRES
Datos de Investigación de INST.DE FISICA DE BUENOS AIRES
Datos de Investigación(IIBBA)
Datos de Investigación de INST.DE INVEST.BIOQUIMICAS DE BS.AS(I)
Datos de Investigación de INST.DE INVEST.BIOQUIMICAS DE BS.AS(I)
Citación
Muraro, Nara Ines; Fernández, Florencia; (2025): Electrophysiological recordings of Drosophila accesory medulla neurons. Consejo Nacional de Investigaciones Científicas y Técnicas. (dataset). http://hdl.handle.net/11336/257796
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