Excitatory Postsynaptic Currents

Simulated traces were generated in NEURON 7.3 with Python 2.7 as interpreter (Hines et al., 2009 (link)). To generate the action potential waveform used for the validation of the slope algorithm, a small current injection (100 pA for 2 s) was injected into a single compartment with a specific membrane capacitance of C_m = 1 μF cm⁻², a leak conductance of 0.1 mS cm⁻² and active sodium (35 mS cm⁻²) and potassium (9 mS cm⁻²) peak conductances, as described by Wang and Buzsáki (1996 (link)).
To validate event detection algorithms, excitatory postsynaptic currents (EPSCs) were generated in a ball-and-stick model with a somatic diameter and length of 20 μm, a dendritic length of 500 μm and a dendritic diameter of 5 μm. Specific membrane capacitance C_m was 1 μF cm⁻², specific membrane resistance R_m was 25 kΩ cm², and specific axial resistivity R_a was 150 Ω cm. Excitatory synaptic conductance changes had a bi-exponential time course with τ_onset = 0.2 ms, τ_decay = 2.5 ms, a peak amplitude of 1 nS and a reversal potential of 0 mV. Dendritic locations of synaptic conductance changes were distributed on the dendrite according to a normal distribution with a center at 400 μm distance from the soma and a standard deviation of 12 μm. Time constants and amplitudes of synaptic conductance changes were varied by multiplying with a random number drawn from a normal distribution with mean 1 and standard deviation 0.3 for time constants and 0.1 for amplitudes. Onset times of synaptic conductance changes were simulated as a Poisson process to yield a mean EPSC frequency of 5 Hz as described by Schmidt-Hieber and Häusser (2013 (link)).

In the figures, original traces show whole-cell patch-clamp recordings obtained from acute brain slice preparations of the hippocampus as described in Schmidt-Hieber et al. (2004 (link)). In Figure 3A, an action potential is evoked by somatic current injection in a granule cell of the dentate gyrus. Figure 3D shows excitatory postsynaptic currents evoked by an action potential between synaptically connected CA3 pyramidal neurons and simultaneous somatic and axonal recordings of an action potential originated at the mossy fiber axon. In Figures 4A,B excitatory postsynaptic currents and corresponding potentials were evoked by a single presynaptic CA3 neuron via recurrent collateral synapses.

Whole-cell patch-clamp recordings were performed on visualized PVN and DMV neurons using an infrared-differential interference contrast (IR/DIC) microscope (BX51WI, Olympus, Japan) with a 40x water-immersion objective. Patch pipettes (3–5 MΩ) were pulled from borosilicate glass capillaries (VitalSense Scientific Instruments Co., Ltd) using a four-stage horizontal micropipette puller (P1000, Sutter Instruments, USA), patch pipettes were filled with intracellular solution (solution components in the Supplementary Data 1) were used for voltage-clamp recording. Signals were amplified with a Multiclamp 700B amplifier, low-pass filtered at 2.8 kHz, digitized at 10 kHz, and recorded in a computer for offline analysis using Clampfit 10.7 software (Molecular Devices) (Zhou et al., 2022 (link)).
The current-evoked firing of PVN^CRH neurons was recorded in current-clamp mode (I = 0 pA). The threshold current of the action potential was defined as the minimum current to elicit an action potential. To visualize the PVN neurons, we injected rAAV-DIO-EYFP into the CRH-Cre mice so that green fluorescent EYFP was expressed only in the CRH neurons.
For validation of chemogenetic virus function. After 3 weeks of chemogenetic virus expression, electrophysiological brain slices were prepared by the above process. The PVN neurons expressing m-Cherry were visualized by using a vertical microscope in Mercury lamp mode, and neuronal responses were recorded before and after CNO administration.
In the vitro electrophysiological recordings of light-evoked response, brain slices were prepared by the above process after 3 weeks of optogenetic virus expression, blue light was delivered through an optical fiber (diameter of 200 μm, Inper) that was positioned 0.2 mm above the surface of the target areas. To characterize the function of rAAV-DIO-ChR2-EYFP in the PVN, ChR2-EYFP⁺ neurons in PVN were visualized by a vertical microscope in Mercury lamp mode, and the responses elicited by different frequencies of blue light stimulation (473 nm, 5–8 mV, pulse width 10 Mm, stimulation frequencies 5 Hz, 10 Hz, 20 Hz) were recorded. For recording light-evoked postsynaptic currents (Fang et al., 2020 (link); Zhou et al., 2022 (link)), DMV expressing ChR2-EYFP⁺ fibers were visualized by a vertical microscope in Mercury lamp mode. The membrane potentials were held at −70 mV for recording the excitatory postsynaptic currents and at 0 mV for recording inhibitory postsynaptic currents, and these recordings were immediately terminated once the series resistance changed more than 10%. To eliminate the polysynaptic components, tetrodotoxin (TTX; 1 μM, Dalian Refine Biochemical Items Co., Ltd.) and 4-aminopyridine (4-AP; 2 mM, Sigma) were added to the standard ACSF to block sodium channels and augment light-induced postsynaptic currents, respectively.

Spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded in the whole-cell voltage-clamp mode. Neurons were held at −70 mV and recordings were obtained in the presence of the sodium channel blocker TTX (1–2 μM), the GABA antagonist,—GBZ (10 μM) and the NMDA antagonist APV (100 μM) in the extracellular solution. For heat treatment, cells were incubated at 40 °C for 1 h and then recorded immediately. Data were acquired by Axon MultiClamp model 700B and Axon Digidata 1440 acquisition systems (Molecular Devices, San Jose, CA, USA) at a at a sampling rate of 20 kHz. mEPSCs were analyzed offline by Clampfit software version 10.6.0.13 (Molecular Devices, San Jose, CA, USA). Statistical significance between groups was assessed by one-way ANOVA followed by specific pairwise comparisons using Student’s t-test, with p values of <0.05 considered statistically significant.