https://www.nature.com/articles/nn.4482
OptogeneticStimulationProcedure(
# In this procedure, adeno-associated viruses encoding cre-dependent
# channelrhodopsin-2 (ChR2) were injected into the nucleus accumbens (NAc) of
# preprodynorphin-IRES-cre mice to express ChR2 in D1 medium spiny neurons. Brain
# slices containing the ventral tegmental area (VTA) were prepared, and
# optogenetic stimulation of NAc→VTA terminals was performed while recording from
# VTA dopamine neurons using whole-cell patch-clamp electrophysiology. The study
# observed that activation of NAc inputs inhibited dopamine neuron firing via
# activation of GABAB receptors, as evidenced by optically evoked inhibitory
# postsynaptic currents that were blocked by GABAB receptor antagonists.
prepare_a_a_v_step=PrepareAAVStep(
# Prepare adeno-associated virus (AAV) encoding cre-dependent
# channelrhodopsin-2 (ChR2) for injection. Ensure the virus is concentrated at
# ~10^12 infectious units/ml.
virus_preparation=VirusPreparationDetails(
virus_type="AAV-DIO-ChR2-YFP",
promoter="EF1α promoter",
concentration="~10^12 infectious units/ml",
injection_volume="500 nl",
injection_rate="100 nl/min",
injection_angle="12°",
injection_coordinates=[
"NAc: +1.7 AP, ±1.6 ML, −4.2 DV",
],
)
),
anesthetize_mice_step=AnesthetizeMiceStep(
# Anesthetize preprodynorphin-IRES-cre mice with 150 mg/kg ketamine and 50
# mg/kg xylazine. Ensure mice are fully anesthetized before proceeding.
chemicals=[
ChemicalAmount(
name="ketamine",
amount="150 mg/kg",
concentration="100 mg/ml",
form="solution",
),
ChemicalAmount(
name="xylazine",
amount="50 mg/kg",
concentration="20 mg/ml",
form="solution",
),
],
mouse_strain="preprodynorphin-IRES-cre",
age="8+ weeks",
sex="either",
monitoring_procedures=[
MonitoringProcedure(
monitoring_type="visual observation",
frequency="continuous",
criteria="loss of righting reflex",
)
],
),
stereotaxic_placement_step=StereotaxicPlacementStep(
# Place the anesthetized mouse in a stereotaxic frame. Maintain the mouse's
# body temperature using a heating pad.
stereotaxic_frame=StereotaxicFrame(model="Standard frame"),
heating_pad=HeatingPad(temperature="37°C", duration="30 minutes"),
),
inject_virus_step=InjectVirusStep(
# Inject 500 nl of AAV-DIO-ChR2-YFP into the nucleus accumbens (NAc) at
# coordinates +1.7 AP, ± 1.6 ML, −4.2 DV, with a 12° angle. Use a 29G
# microinjection needle connected to a Hamilton syringe. Allow the virus to
# diffuse for 5 minutes post-injection.
injection_details=InjectionDetails(
injection_volume="500 nl",
injection_duration="5 min",
injection_coordinates="AP: +1.7, ML: ±1.6, DV: −4.2",
injection_angle="12°",
microinjection_needle_gauge="29G",
syringe_type="Hamilton syringe",
),
virus_diffusion_time="5 min",
virus_concentration="~10^12 infectious units/ml",
virus_promoter="EF1α promoter",
cre_line="Dyn-cre",
),
monitor_recovery_step=MonitorRecoveryStep(
# Allow the mice to recover from surgery and express ChR2 for 3‐8 weeks.
# Monitor mice for any signs of distress or infection during the recovery
# period.
recovery_duration="3-8 weeks",
monitoring_frequency="Daily",
signs_of_distress=["Lethargy", "Weight loss", "Poor grooming"],
signs_of_infection=["Swelling", "Redness", "Discharge"],
documentation_method="Observation logs",
),
anesthetize_and_perfuse_step=AnesthetizeAndPerfuseStep(
# Anesthetize mice with Euthasol and perfuse with ice-cold artificial
# cerebrospinal fluid (ACSF) to preserve brain tissue. Ensure perfusion is
# complete before proceeding.
anesthesia=Anesthesia(
type="Euthasol",
dose="150 mg/kg ketamine, 50 mg/kg xylazine",
duration="Until fully anesthetized",
),
perfusion=Perfusion(
solution="ice-cold ACSF",
duration="Until complete",
temperature="0 °C",
substitution="NMDG for sodium",
acsf_composition=[
"92 mM NMDG",
"20 mM HEPES",
"25 mM glucose",
"30 mM NaHCO3",
"1.2 mM sodium phosphate",
"2.5 mM KCl",
"5 mM sodium ascorbate",
"3 mM sodium pyruvate",
"2 mM thiourea",
"10 mM Mg-sulfate",
"0.5 mM CaCl2",
],
),
),
prepare_brain_slices_step=PrepareBrainSlicesStep(
# Prepare horizontal brain slices (150-200 µm thick) containing the VTA using
# a vibratome. Use ACSF with NMDG substituted for sodium during slicing to
# maintain neuronal health.
vibratome_model="VT-1200",
section_thickness="150-200 µm",
acsf_solution=ACSFDetails(
composition=[
"92 mM NMDG",
"20 mM HEPES",
"25 mM glucose",
"30 mM NaHCO3",
"1.2 mM sodium phosphate",
"2.5 mM KCl",
"5 mM sodium ascorbate",
"3 mM sodium pyruvate",
"2 mM thiourea",
"10 mM Mg-sulfate",
"0.5 mM CaCl2",
],
ph="7.35",
osmolarity="~305 mOsm",
bubbling_gas_composition="95% O2, 5% CO2",
),
slicing_temperature="Ice-cold",
),
incubate_brain_slices_step=IncubateBrainSlicesStep(
# Incubate brain slices in ACSF at 32–34 °C for at least 1 hour. Ensure slices
# are adequately oxygenated by bubbling ACSF with 95% O2 and 5% CO2.
incubation_temperature=Temperature(degrees_celsius="32–34"),
incubation_duration="1 hour",
oxygenation_gas_composition="95% O2 and 5% CO2",
),
position_optical_fiber_step=PositionOpticalFiberStep(
# Position a 200-µm optical fiber coupled to a diode-pumped solid-state laser
# above the brain slice, targeting the recorded VTA dopamine neuron.
optical_fiber=OpticalFiber(
core_diameter="200 µm",
wavelength="473 nm",
intensity="2–5 mW",
pulse_duration="3 ms",
frequency="20 Hz",
),
laser=Laser(
type="diode-pumped solid-state laser",
wavelength="473 nm",
pulse_duration="3 ms",
intensity="2–5 mW",
),
neuron_target=NeuronTarget(
neuron_type="dopamine neurons",
location="lateral VTA",
target_area="caudal VTA and substantia nigra",
),
positioning_details=PositioningDetails(
distance_to_slice="200 µm", angle="0°", position="above the slice"
),
),
whole_cell_patch_clamp_step=WholeCellPatchClampStep(
# Perform whole-cell patch-clamp recordings on VTA dopamine neurons. Use patch
# pipettes filled with internal solution containing potassium methylsulfate,
# NaCl, MgCl2, BAPTA, sodium phosphocreatine, Mg-ATP, and Na2-GTP.
internal_solution=InternalSolution(
components=[
"115 mM potassium methylsulfate",
"20 mM NaCl",
"1.5 mM MgCl2",
"10 mM BAPTA",
"10 mM sodium phosphocreatine",
"4 mM Mg-ATP",
"0.4 mM Na2-GTP",
],
pH="7.35",
osmolarity="285 mOsm",
),
target_neurons=["dopamine neurons"],
recording_chamber="Superfused with 32–34 °C ACSF",
amplifier=Amplifier(
model="MultiClamp 700B",
software="pClamp 10.3",
series_resistance_monitoring="Maintained below 30 MΩ",
),
voltage_clamp_conditions="Voltage clamped at −55 mV",
neuron_identification_criteria=[
"long action potential width",
"low tonic firing rates",
"h-current",
"TH immunoreactivity",
],
recording_temperature="32–34 °C",
optical_stimulation_protocol="20 pulses, 20 Hz, 473-nm, 2–5 mW, 3 ms",
),
optogenetic_stimulation_step=OptogeneticStimulationStep(
# Optogenetically stimulate NAc→VTA terminals with 473-nm wavelength light
# (2–5 mW, 20 Hz, 3 ms pulses) while voltage clamping neurons at −55 mV to
# evoke GABAB responses.
light_stimulation=LightStimulation(
wavelength="473 nm",
power="2-5 mW",
frequency="20 Hz",
pulse_duration="3 ms",
number_of_pulses="20",
),
voltage_clamp=VoltageClamp(
holding_potential="-55 mV",
series_resistance="< 30 MΩ",
clamp_type="Voltage",
),
),
record_o_i_p_s_c_step=RecordOIPSCStep(
# Record optically evoked inhibitory postsynaptic currents (oIPSCs) in
# dopamine neurons. Monitor and maintain series resistance below 30 MΩ
# throughout the recording.
recording_parameters=WholeCellRecordingParameters(
amplifier="MultiClamp 700B",
software="pClamp 10.3",
series_resistance_threshold="30 MΩ",
monitoring_frequency="every 30 s",
resistance_change_threshold="20%",
holding_current="0 pA",
voltage_clamp_protocol="Voltage clamp at −55 mV",
patch_clamp_technique="Whole-cell patch-clamp",
electrophysiological_characteristics="Long action potential width, low tonic firing rate, presence of h-current, TH immunoreactivity",
internal_solution_composition=[
"115 mM potassium methylsulfate",
"20 mM NaCl",
"1.5 mM MgCl2",
"10 mM BAPTA",
"10 mM sodium phosphocreatine",
"4 mM Mg-ATP",
"0.4 mM Na2-GTP",
],
internal_solution_ph="7.35",
internal_solution_osmolarity="285 mOsm",
series_resistance_monitoring_method="Monitored continuously, excluded if changed >20%",
reference_electrode_solution=[
"125 mM NaCl",
"2.5 mM KCl",
"1.25 mM NaH2PO4",
],
action_potential_width=">1.1 ms",
tonic_firing_rate="1-4 Hz",
h_current_amplitude="≥70 pA",
th_immunoreactivity="Present",
),
optical_stimulation_protocol=OpticalStimulationProtocol(
wavelength="473 nm",
power="2–5 mW",
duration="3 ms",
frequency="20 Hz",
pulse_count="20",
stimulation_device="Diode-pumped solid-state laser",
),
neuron_identification_method="Morphology, electrophysiology, TH immunoreactivity",
recording_temperature="32–34 °C",
acsf_composition=[
"125 mM NaCl",
"2.5 mM KCl",
"1.25 mM sodium phosphate",
"1 mM MgCl2",
"2.4 mM CaCl2",
"26 mM sodium bicarbonate",
"11 mM glucose",
],
),
apply_g_a_b_a_b_antagonist_step=ApplyGABABAntagonistStep(
# Apply GABAB receptor antagonist (e.g., CGP 35348) to confirm that oIPSCs are
# mediated by GABAB receptors. Observe for complete blockade of oIPSCs to
# verify GABAB receptor involvement.
antagonist=Chemical(name="CGP 35348"),
observation="Complete blockade of oIPSCs confirms GABAB receptor involvement.",
),
document_observations_step=DocumentObservationsStep(
# Document all observations and data, including the amplitude and frequency of
# oIPSCs, using appropriate software for analysis. Ensure data is backed up
# and stored securely.
observations=[
"Optically evoked IPSCs in dopamine neurons",
"Amplitude and frequency of oIPSCs",
],
data_analysis_software="GraphPad Prism",
secure_storage_location="NIDA secure server",
backup_procedures="Daily backups on NIDA server",
analysis_parameters=[
"Amplitude and frequency of oIPSCs",
"Blocked by CGP 35348",
"Measured at -55 mV",
],
),
)