Electrical synapses between coupled neurons are typically characterized by coupling coefficients, which are measured by delivery of hyperpolarizing current pulses to one neuron that result in voltage responses in both neurons. This technique requires dual whole-cell patch recordings. Here, we developed and used opto-dL, which measures electrical synapses by the change in action potential latency to rheobase stimuli in one recorded neuron induced by simultaneous focal photoexcitation of soma-targeted opsins expressed in a potentially coupled neighbor. This technique requires only a single recording and can measure synapses from multiple coupled partners rapidly in sequence during a single recording. Our use of opto-dL enabled detection and measurement of electrical synapses in mature rodent thalamic reticular nucleus, where myelination of fibers after eye opening occludes paired recordings. Because dL requires recording from only one neuron, it can be used to map electrically coupled networks.

We first measured both coupling coefficients and dL in a population of pairs of neurons and in connexin36 knockdown tissue to validate opto-dL. We then measured electrical synapses and mapped networks in mature thalamic reticular nucleus and somatosensory cortex.

This dataset comprises electrophysiological recordings from single and pairs of cells in the thalamic reticular nucleus and somatosensory cortex during photoexcitation of neighboring neurons.

Animals

Experiments were approved by the Lehigh University IACUC. SOM-Cre (Jax: 013044) or PV-Cre (Jax: 008069) mice were used for experiments. Mice were at least P28 for surgeries and P42 for electrophysiological recordings. For one juvenile experiment (Fig. 1), a calbindin-Cre (Jax: 028532) mouse was injected at P0 and electrophysiological recordings occurred at P14. To target our CRISPR knock down selectively to the PV-expressing GABAergic neurons we crossed male Rosa26-lox-stop-lox-Cas9/GFP mice (Jax: 026175) with PV-Cre (Jax: 017320), generating Cas9 and GFP reporter expression in PV neurons (PV-Cre-CRISPRCas9-GFP).

Electrophysiology

Mice were transcardially perfused with NMDG ACSF solution (in mM): 92 NMDG, 2.5 KCl, 1.25 NaH₂PO₄, 30 NAHCO₃, 20 HEPES, 25 glucose, 2 thiourea, 5 NA ascorbate, 3 NA pyruvate, 0.5 CaCl₂, and 10 MgSO₄·7H₂O (315 mOsm L⁻¹, 7.3-7.4 pH). Horizontal brain slices 250 µm thick were cut and incubated in NMDG solution. Slices were incubated at 34°C for 12-15 min in NMDG following cutting and returned to HEPES ACSF at room temperature until recording. The HEPES ACSF solution comprised in mM: 92 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 glucose, 2 Thiourea, Na ascorbate, NA pyruvate, 2 CaCl2, and 2 MgSO₄·7H₂0 (315 mOsm L⁻¹, 7.3-7.4 pH). The ACSF bath during recording contained (in mM): 126 NaCl, 3 KCl, 1.25 NaH₂PO₄, 2 MgSO₄, 26 NaHCO₃, 10 dextrose, and 2 CaCl₂ (315–320 mOsm L⁻¹, saturated with 95% O₂/5% CO₂). The submersion recording chamber was held at 34°C (TC-324B, Warner Instruments). Electrodes were filled with (in mM): 135 potassium gluconate, 2 KCl, 4 NaCl, 10 Hepes, 0.2 EGTA, 4 ATP-Mg, 0.3 GTP-Tris, and 10 phosphocreatine-Tris (pH 7.25, 295 mOsm L⁻¹). 1 M KOH was used to adjust pH of the internal solution. The approximate bath flowrate was 2 ml min⁻¹ and the recording chamber held approximately 5 ml solution.

The TRN was visualized under 4x magnification, and single or pairs of adjacent TRN cells with strong ChroME-mRuby expression nearby but without preference to core or shell or sensory sector were identified and patched under 40x IR-DIC optics (SliceScope, Scientifica, Uckfield, UK). Cell type was determined by mRuby epifluorescence in the patched neurons. Cells with low fluorescent contrast were avoided. The mRuby reporter was excited by a 560 nm diode delivered through the objective (CoolLED pE-300). Voltage signals were amplified and lowpass filtered at 8 kHz (MultiClamp, Axon Instruments, Molecular Devices, Sunnyvale, CA, USA), digitized at 20 kHz with custom Matlab routines controlling a National Instruments (Austin, TX, USA, USB6221 DAQ board), and data were stored for offline analysis in Matlab (Mathworks, R2023b, Natick, MA, USA). Recordings were made in whole-cell current-clamp mode. Negative current was used to maintain cells at −70 mV to -60 mV during recordings. Pipette resistances were 5 - 9 MΩ before bridge balance; recordings were discarded if access resistance exceeded 25 MΩ. Voltages are reported uncorrected for the liquid junction potential.

Stereotaxic surgery

SOM-Cre (Jax: 013044), PV-Cre (Jax: 008069), or PV-Cre-CRISPR-Cas9-GFP mice were anesthetized via inhaled isoflurane (0.8-1.2%) and 0.1 mL of 0.015 mg/mL buprenorphine via intraparietal injection. Mice were positioned on a heated pad and in a stereotax (Kopf). After exposure of the skull, a dental drill was used to open a hole above the injection site. Then, AAV9-CAG-DIO-ChroME-st-P2A-H2B-mRuby (titer 2.7 x 10¹³ GC/mL) was injected at a rate of 100 nL/min (WPI UMP-3). pAAV-CAG-DIO-ChroME-ST-P2A-H2B-mRuby3 was a gift from Hillel Adesnik (Addgene viral prep # 108912-AAV9; http://n2t.net/addgene:108912; RRID:Addgene_108912). Viral vectors used for knocking down Cx36 or Cx29 were designed by David Uygun and Radhika Basheer as described previously. Briefly, the PX552 plasmid (Addgene plasmid #60958) was modified to encode triple U6-sgRNA cassettes each targeting a distinct locus of the target genes. The GFP sequence was replaced by the sequence encoding the blue fluorescent protein (BFP). The coordinates for injections were -1.35 AP, ± 2.1 ML, -3.0 and -3.2 DV for TRN and -0.5 AP, ± 3.0 ML, -2.0 and -2.6 DV for somatosensory cortex. We injected a total of 600 nL of virus for TRN and 800 nL of virus for somatosensory cortex. Post surgery and scalp suturing, mice were given an intraperitoneal injection of 0.1 mL of 0.5 mg/mL carprofen and 1 mL saline, then monitored for recovery. Mice were given at least two weeks for viral expression before experiments. For the juvenile surgery, a calbindin-Cre (Jax: 028532) mouse was anesthetized via hypothermia on a cold metal plate, and the coordinates for injection targeting TRN were -1.4 AP, ± 1.9 ML, -1.4 and -1.9 DV.

Image processing

All fluorescence images were modified in Fiji and pseudocolored to facilitate visualization. All infra-red differential interference contrast images were flat-field corrected.

Opto-ẟL

For the rheobase current necessary to measure ẟL, we used the smallest stimuli that drove spiking at least 80% of the time, with the CV of latency less than 0.2. Trials were excluded if latency was less than 20 ms for rheobase stimuli.

To perform single-cell optogenetic stimulation, we used a 560 nm LED (Mightex) and digital mirror device (Mightex Polygon 1000) to direct light to an area the size of an individual soma (7 - 8 µm diameter circle). The area used covered the entire soma without extending beyond the edge of the soma. Prior to any experiments, we patched an opsin-expressing cell to determine an intensity of light that was sufficient to drive bursts for a 50 ms stimulus, and we then used a photostimulus that was 50% stronger for all subsequent experiments in that slice. Light intensity ranged from 3.7 - 99.8 mW/mm² with most experiments occurring in 6.2 - 25.0 mW/mm² range.

Single-cell optogenetic stimulation is confounded by off-target excitation by light scatter for both 1 photon and 2 photon stimulation paradigms. For the purposes of mapping synaptic connections, scatter-induced photostimulation of the patched neuron is a potential confound. Depolarization from photostimulation caused by light scatter is directly proportional to the distance between the patched neuron and focal stimulation (Supp. Fig. 3). Opto-δL is well-suited to overcome the problem of unintended photostimulation to the patched neuron because the method quantifies latency of spikes rather than voltage changes. Consequently, comparing latency when a nearby neuron is photostimulated to latency when an equidistant empty control location is photostimulated allowed us to distinguish the contribution of GJs to spike times.

After patching TRN neurons we used one of two methods to make measurements that would control for the effects of light scattering and then to compute δL:

i) Direct corrections to δL: We delivered a rheobase stimulus to the patched neuron alone 10 times. Interleaved between those stimulations, we gave the neuron a rheobase stimulus while also focally photostimulating either a nearby mRuby⁺ neuron or an equidistant empty spot. Offline, we measured the latency of spikes from stimulation onset for each condition. We report ẟL as the change in latency induced by focal photostimulation of the nearby neuron minus the change in latency induced by photostimulation of an empty area.

ii) Correction to Iapp: We applied focal photostimulation ten times to an empty location equidistant from the patched neuron as the nearby neuron of interest. We measured the average depolarizing current that resulted from the light scattering. We then adjusted rheobase Iapp by average current caused by light scatter (Supp. Fig. 4) and used that adjusted Iapp when photostimulating the nearby mRuby⁺ or an equidistant empty location. Offline, we calculated ẟL by comparing latency changes between when light targeted the empty control location ( ) compared to light targeting the mRuby⁺ neuron ( ).

We directly corrected δL for 27 homocellular network measurements, and we used correction to I_app for 51 homocellular network measurements.

Numerical analysis and statistics

To calculate δL we used the average spike latency from control trials (t₁) and average spike latency when a nearby neuron was photostimulated (t₂):

We used a Friedman’s test on the sets of latencies during rheobase-only, rheobase with blank spot photostimulation, and rheobase with neighbor photostimulation for homocellular networks that used direct correction by blank spot stimulation, and then used a Wilcoxon signed-rank test for post-hoc pairwise comparisons of spike latencies. We used a Wilcoxon signed-rank test to test latencies in experiments corrected by I_app. We used the Mann-Whitney U test for comparisons between neuron types, and we used Fisher’s exact tests to compare coupling rate and electrical synapses counts per network. Statistical tests were considered significant when p < 0.05.

Networks: In our analysis of network size, we required three or more connections tested from a hub cell to be considered as a network.