Long-range inhibition synchronizes and updates prefrontal task activity – Nature

Mice

All animal care, procedures and experiments were conducted in accordance with NIH guidelines and approved by the Administrative Panels on Laboratory Animal Care at the University of California, San Francisco as well as followed French and European guidelines for animal experimentation and in compliance with the institutional animal welfare guidelines of the Paris Brain Institute. Mice were group housed (2–5 siblings) in a temperature-controlled environment (22–24 °C), had ad libitum access to food and water, and reared in normal lighting conditions (12-h light-dark cycle), until rule-shift experiments began. All experiments were done using PV-cre, wild-type C57/Bl6, and PV-cre Ai14 lines (The Jackson Laboratory). Both male and female adult mice (8–10 weeks and 10–20 weeks old at time of experiment) were used for slice electrophysiology and behavioural experiments, respectively.

Surgery

Male and female mice were anaesthetized using isoflurane (2.5% induction, 1.2–1.5% maintenance, in 95% oxygen) and placed in a stereotaxic frame (David Kopf Instruments). Body temperature was maintained using a heating pad. An incision was made to expose the skull for stereotaxic alignment using bregma and lambda as vertical references. The scalp and periosteum were removed from the dorsal surface of the skull and scored with a scalpel to improve implant adhesion. Viruses were infused at 100–150 nl min⁻¹ through a 35-gauge, beveled injection needle (World Precision Instruments) using a microsyringe pump (World Precision Instruments, UMP3 UltraMicroPump). After infusion, the needle was kept at the injection site for 5–10 min and then slowly withdrawn. After surgery, mice were allowed to recover until ambulatory on a heated pad, then returned to their home cage.

For slice electrophysiology experiments using optogenetic opsins, mice were injected unilaterally in the mPFC, near the border between the prelimbic and infralimbic cortices (1.7 anterior-posterior (AP), + or −0.3 mediolateral (ML), and −2.75 dorsoventral (DV) mm relative to bregma) with 0.4 μl of AAV5-EF1α-DIO-ChR2-eYFP (UNC Vector Core) to selectively target neurons expressing Cre. To allow for virus expression, experiments began at least 3 weeks after injection. For retrograde labelling of mPFC neurons that project callosally versus to mediodorsal (MD) thalamus, cholera toxin subunit B (CTb) conjugated with Alexa Fluor 488 (CTb-488, Invitrogen; 0.2% w/v, 400 nl) was injected in the PFC ipsilateral to the previous AAV-DIO-ChR2 injection (1.7 AP, −0.4 ML, and −2.5 DV mm relative to bregma) and Alexa Fluor 594-conjugated CTb (CTb-594, Invitrogen; 0.2% w/v, 400 nl) was injected in the MD thalamus (contralateral to the AAV-DIO-ChR2 injection) (−1.7 AP, +0.35 ML, −3.5 DV mm relative to bregma). For retrograde labelling of mPFC neurons projecting to the NAc versus dorsal striatum, CTb-488 (0.2% w/v, 400 nl) was injected in the NAc (contralateral to the AAV-DIO-ChR2 injection) (1.34 AP, −1.0 ML, and −4.6 DV mm relative to bregma) and CTb-594 (0.2% w/v, 600 nl) was injected in the dorsal striatum (also contralateral to the AAV-DIO-ChR2 injection) (+0.9 AP, +1.16 ML, −3.0 DV mm relative to bregma). To allow time for retrograde transport of CTb, experiments began one week after CTb injection.

For behavioural experiments using eNpHR and control optogenetics, mice were injected unilaterally in the mPFC, near the border between the prelimbic and infralimbic cortices (1.7 AP, + or −0.3 ML, and −2.75 DV mm relative to bregma) with 1 μl of AAV2-EF1α-DIO-eNpHR3.0-mCherry (UNC Vector Core), 1 μl of AAV5-EF1α-DIO-eYFP (UNC Vector Core), AAV5-hSynapsin-eNpHR3.0-eYFP (UNC Vector Core) or AAV5-hSynapsin-tdTomato (UNC Vector Core), to selectively target Cre-expressing cells or non-selectively target prefrontal neurons. After injecting virus, a 200/240 μm (core/outer) diameter, NA = 0.22, mono fibre-optic cannula (Doric Lenses, MFC_200/240-0.22_2.3mm_FLT) was slowly inserted into mPFC until the tip of the fibre reached a DV depth of −2.25. Implants were affixed onto the skull using Metabond Quick Adhesive Cement (Parkell). To allow for virus expression, behavioural experiments began at least five weeks after injection.

For behavioural experiments using both NpHR and ChR2 optogenetics, mice were injected unilaterally in the mPFC, near the border between the prelimbic and infralimbic cortices (1.7 AP, −0.3 ML, and −2.75 DV mm relative to bregma) with 0.75 μl of AAV8-nEF-Con/Fon-NpHR3.3-eYFP (Addgene) and 0.75 μl of AAV8-nEF-Con/Fon-ChR2-mCherry (Addgene) or 0.75 μl of AAV8-EF1α-Con/Fon-mCherry (Addgene) and contralaterally in PFC (1.7 AP, +0.3 ML, −2.75 DV mm relative to bregma) with 0.7 μl AAVrg-EF1α-FlpO (Addgene), to selectively target Cre-expressing cells in ipsilateral PFC that send projections to contralateral PFC. After injecting virus, a 200/240 μm (core/outer) diameter, NA = 0.22, mono fibre-optic cannula (Doric Lenses, MFC_200/240-0.22_2.3mm_FLT) was slowly inserted into mPFC until the tip of the fibre reached a DV depth of −2.25. Implants were affixed onto the skull using Metabond Quick Adhesive Cement (Parkell). To allow for virus expression, behavioural experiments began at least five weeks after injection.

For behavioural experiments that combined dual-site voltage indicator imaging with optogenetics, mice were injected bilaterally at 3 depths (DV: −2.5, −2.25 and −2.0) at the following AP/ML for mPFC: 1.7 AP, ±0.3 ML with 3 × 0.2 μl of AAV1-CAG-DIO-Ace2N-4AA-mNeon (Virovek). Mice were also injected unilaterally in the mPFC (1.7 AP, −0.3 ML, and −2.75 DV mm relative to bregma) with either 1 μl of AAV2-EF1α-DIO-eNpHR3.0-BFP (Virovek) or 1 μl of AAV5-EF1α-DIO-mCherry (UNC Vector Core), 1 μl of AAV2-hSynapsin-eNpHR3.0-mCherry (UNC Vector Core) or 1 μl of AAV2-hSynapsin-mCherry (UNC Vector Core). After injection of virus, two 400/430 μm (core/outer) diameter, NA = 0.48, multimode fibre implants (Doric Lenses, MFC_400/430-0.48_2.8mm_ZF1.25_FLT) were slowly inserted into the mPFC at a ±12° angle using the following coordinates: 1.7 (AP), ±0.76 (ML), −2.14 (DV). To allow for virus expression, behavioural experiments began at least five weeks after injection.

For behavioural experiments that combined in vivo calcium imaging with optogenetics, mice were injected unilaterally at 4 depths (DV: −2.75, −2.5, −2.25, −2.0) at the following AP/ML for mPFC: 1.7 AP, +0.4 ML with diluted (1:3; Addgene) 4 × 0.15 μl of AAV9-synapsin-jGCaMP7f-WPRE (Addgene). Mice were also injected unilaterally in the contralateral mPFC (1.7 AP, −0.3 ML, and −2.75 DV mm relative to bregma) with either 1 μl of AAV2-EF1α-DIO-eNpHR3.0-mCherry (UNC Virus Core), 1 μl of AAV5-EF1α-DIO-mCherry (UNC Virus Core), 1 μl of AAV2-hSynapsin-eNpHR3.0-mCherry (UNC Virus Core) or 1 μl of AAV2-hSynapsin-mCherry (UNC Virus Core). After injection of virus, a 0.5 mm × 4.0 mm-long integrated GRIN lens (Inscopix) was slowly advanced into the mPFC until the tip was placed at 1.7 AP, +0.4 ML, DV −2.25 and cemented in place with Metabond Quick Adhesive Cement (Parkell). To allow for virus expression, behavioural experiments began at least five weeks after injection.

Slice preparation and analysis

Adult mice were anaesthetized using isoflurane and decapitated, and their brains were rapidly removed. Coronal slices (250 μm thick) were cut from adult mice of either sex using a vibratome (VT1200S Leica Microsystems) and a chilled slicing solution in which Na⁺ was replaced by sucrose, then incubated in warmed artificial cerebrospinal fluid (ACSF) at 30-31 °C for 15 min and then at least 1 h at room temperature before being used for recordings. ACSF contained (in mM): 126 NaCl, 26 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl and 10 glucose. Slices were secured by placing a harp along the midline between the two hemispheres.

Somatic whole-cell patch recordings were obtained from ChR2-negative neurons amid ChR2-positive axonal fibres in the mPFC contralateral to the site of virus injection, on an upright microscope (BX51WI; Olympus). Recordings were made using a Multiclamp 700A (Molecular Devices). Patch electrodes (tip resistance = 2–6 MOhms) were filled with the following (in mM): 130 potassium gluconate, 10 KCl, 10 HEPES, 10 EGTA, 2 MgCl₂, 2 MgATP, and 0.3 NaGTP (pH adjusted to 7.3 with KOH). All recordings were at 32.5 ± 1 °C. Series resistance was usually 10–20 MΩ, and experiments were discontinued above 30 MΩ.

Intrinsic properties were calculated based on the current-clamp responses to a series of 250 msec current pulse injections from −200 to 450 pA (50 pA per increment). Spiking properties were calculated based on the response to a current pulse that was 100 pA above the minimal level that elicited spiking. Neurons were classified as fast spiking if they met 3 out of the 4 following criteria: action potential (AP) half-width was < 0.5 ms, firing frequency > 50 Hz, fast after hyperpolarization (fAHP) amplitude > 14 mV, and spike-frequency accommodation (SFA) index < 2.

In vitro ChR2 stimulation

Stimulation of channelrhodopsin (ChR2) in callosal PV terminals was performed using ~4–5 mW flashes of light generated by a Lambda DG-4 high-speed optical switch with a 300 W Xenon lamp (Sutter Instruments) or by an LED (Cairn Research OptoLED Lite), and an excitation filter set centred around 470 nm, delivered to the slice through a 40× objective (Olympus). Illumination was delivered across a full high-power (40×) field. To measure IPSPs, current-clamp recordings were performed while stimulating ChR2 using trains of light pulses (5 ms light pulses at 5 Hz, 20 Hz, and 40 Hz). In experiments in which glutamatergic and GABA_A receptors were blocked, drugs were bath applied at the following concentrations (in μM): 10 CNQX (Tocris) or 20 DNQX (Tocris), 50 APV (Tocris), and 10 gabazine (Sigma). Drugs were prepared as concentrated stock solutions and were diluted in ACSF on the day of the experiment.

Rule-shift task

This cognitive flexibility task has been described previously⁶. In brief, mice are singly housed and habituated to a reverse light/dark cycle, and food intake is restricted until the mouse is 80–85% of the ad libitum feeding weight. After mice reached their target weight, they underwent one day of habituation. On this day, mice were given ten consecutive trials with the baited food bowl to ascertain that they could reliably dig and that only one bowl contained food reward. All mice were able to dig for the reward, and started the task the next day. At the start of each trial, the mouse was placed in its home cage to explore two bowls, each containing one odour and one digging medium, until it dug in one bowl, signifying a choice. As soon as a mouse began to dig in the incorrect bowl, the other bowl was removed, so there was no opportunity for ‘bowl switching’ (digging is defined as the sustained displacement of the medium within a bowl). The bait was a piece of a peanut butter chip (approximately 5–10 mg in weight) and the cues, either olfactory (odour) or somatosensory and visual (texture of the digging medium which hides the bait), were altered and counterbalanced. All cues were presented in small animal food bowls (All Living Things Nibble bowls, PetSmart) that were identical in colour and size. Digging media were mixed with the odour (0.01% by volume) and peanut butter chip powder (0.1% by volume). All odours were ground dried spices (McCormick or Alpi Nature garlic and McCormick or Albert Ménès coriander), and unscented digging medium (Mosser Lee White Sand Soil Cover or Scalare Sable de Rivière, Natural Integrity Clumping Clay or Monoprix cat litter).

After mice reached their target weight, they underwent one day of habituation. On this day, mice were given ten consecutive trials with the baited food bowl to ascertain that they could reliably dig and that only one bowl contained food reward. Specifically, the habituation trials are used to train the mouse on the mechanics of the task, there is no association made between food reward and cue. All mice were able to dig for the reward. Mice do not undergo any other specific training before being tested on the task. Then, on days 1 and 2 (and in some cases, on additional days as well), mice performed the task (this was the testing done for experiments). After the task was done for the day, the bowls were filled with different odour–medium combinations and food was evenly distributed among these bowls and given to the mouse so that no specific cue was rewarded greater than the other cues present. The same stimuli were used across days—only the cue that is associated with the food reward changed.

Mice were tested through a series of trials. The determination of which odour and medium to pair and which side (left or right) contained the baited bowl was randomized (subject to the requirement that the same combination of pairing and side did not repeat on more than three consecutive trials) using https://www.random.org. On each trial, while the particular odour–medium combination present in each of the two bowls may have changed, the particular stimulus (for example, a particular odour or medium) that signalled the presence of food reward remained constant over each portion of the task (initial association and rule shift). If the initial association paired a specific odour with food reward, then the digging medium would be considered the irrelevant dimension. The mouse is considered to have learned the initial association between stimulus and reward if it makes eight correct choices during ten consecutive trials. Each portion of the task ended when the mouse met this criterion. Following the initial association, the rule-shift portion of the task began, and the particular stimulus associated with reward underwent an extra-dimensional shift. For example, if an odour had been associated with reward during the initial association, then a digging medium was associated with reward during the rule-shift portion of the task. The mouse is considered to have learned this extra-dimensional rule shift if it makes 8 correct choices during ten consecutive trials. When a mouse makes a correct choice on a trial, it is allowed to consume the food reward before the next trial. Following correct trials, the mouse is transferred from the home cage to a holding cage for about 10 s while the new bowls were set up (ITI). After making an error on a trial, a mouse was transferred to the holding cage for about 2 min (ITI). For Extended Data Fig. 4p–z, the ITI following errors was 30 s in the holding cage. All animals performed the initial association in a similar number of trials (average: 10–15 trials). Experiments were performed blind to the virus injected. Videos were manually scored with a temporal resolution of 1 s.

For analyses (described below), the onset of digging was chosen as the time of a decision for two reasons. First, as noted above, once a mouse began to dig in the incorrect bowl, the other (correct) bowl was removed. Second, only upon the commencement of digging could a mouse determine whether reward was present in the chosen bowl and obtain feedback about whether or not it had made a correct choice. The time windows used for analysis excluded periods when the mouse moved from the home cage to the holding cage and vice versa.

Rule-reversal task

This cognitive flexibility task was described previously^6,8. Similarly to the mechanics of the rule-shift task described above, after the initial association, the rule-reversal portion of the task began, and the particular stimulus associated with reward underwent an intra-dimensional reversal. For example, if an odour had been associated with reward during the initial association, then the previously unrewarded odour became associated with reward during the rule-reversal portion of the task. The mouse was considered to have learned the intra-dimensional rule reversal when it made eight correct choices out of ten consecutive trials.

In vivo optogenetic stimulation

For behavioural experiments using optogenetic eNpHR stimulation: A 532 nm green laser (OEM Laser Systems) was coupled to the mono fibre-optic cannula (Doric Lenses) with a zirconia sleeve (Doric Lenses) through a 200-μm-diameter mono fibre-optic patch cord (Doric Lenses) and adjusted such that the final light power was 2.5 mW. For behavioural experiments using optogenetic ChR2 stimulation: A 473 nm blue laser (OEM Laser Systems) was coupled to the mono fibre-optic cannula (Doric Lenses) with a zirconia sleeve (Doric Lenses) through a 200 μm diameter mono fibre-optic patch cord (Doric Lenses, Inc.) and adjusted such that the final light power was 0.5 mW. A function generator (Agilent 33500B Series Waveform Generator) connected to the laser generated a 40-Hz train of 5-ms pulses.

Extended Data Fig. 2 shows experiments designed to control for potential behavioural effects of scattered light from one hemisphere activating eNpHR in PV neuron cell bodies in the contralateral hemisphere. These experiments used a final light power of 0.1 mW when connected to the 532 nm green laser (OEM Laser Systems) or 638 nm red laser (Doric Lenses). To determine the appropriate light power for these experiments, a mouse was implanted with a dual fibre-optic cannula (Doric Lenses; DFC_200/240-0.22_2.3mm_GS0.7_FLT) without virus injection in order to measure light scattering from one mPFC to the contralateral hemisphere. Using a dual fibre-optic patch cord (Doric Lenses; DFP_200/240/900-0.22_2m_GS0.7-2FC), light was delivered to the mPFC on one side, and the light coming through the other side was measured using a light meter (ThorLabs, PM100D). The final light power delivered to one mPFC was 2.5 mW, across wavelengths 532 nm, 594 nm, and 638 nm—similar to what was used in the optogenetic and optogenetic and dual-site voltage indicator experiments. Measurements of 40 nW, 20 nW, and 50 nW at the contralateral fibre were observed, respectively. Accounting for transmission loss of the patch cord (for example, only 80% of the light is transmitted from end to end), the scattered light power entering the fibre tip would be 50 nW, 25 nW and 62.5 nW respectively. This measurement likely overestimates the actual light entering via the fibre tip located in brain parenchyma since it will include both scattered light within the brain and contamination from ambient room light. Conversely, this measurement only includes light located in the vicinity of the fibre tip that is traveling at an appropriate angle to enter the fibre (which has numerical aperture 0.22 implying a 25.4° acceptance angle). Therefore, to be extremely conservative, experiments in Extended Data Fig. 2 utilized a final light power of 0.1 mW, which is >1,000 times stronger than the measured scattered light power. Both 532 nm and 638 nm at this final light power ipsilateral to the viral injection site was used.

Experiments in Extended Data Fig. 2l–o were similar to other experiments using optogenetic eNpHR stimulation except in the following respects: mice were injected with 0.7 μl of AAV2-EF1α-DIO-eNpHR3.0-mCherry (UNC Vector Core) and 0.4 μl of AAV5-EF1α-DIO-ChR2-eYFP (UNC Vector Core), to selectively target Cre-expressing cells (although ChR2 stimulation was not ultimately used in these experiments). Light for optogenetic eNpHR stimulation was specifically switched on on day 2 during ITIs (when the mouse was in the holding cage) and switched off during the trial in the home cage.

For all optogenetic experiments, light stimulation began once mice reached the 80% criterion during the initial association portion of the task. Mice then performed three additional initial association trials with the light stimulation before the rule-shift portion of the task began. The light stimulation did not alter the performance or behaviour of the mice during these three extra trials of the initial association. Experiments were performed blind to virus injected.

Combined dual-site voltage indicator imaging and optogenetic eNpHR stimulation

High-bandwidth, time-varying bulk fluorescence signals were measured at each recording site using the dual-site voltage-indicator technique⁸, with some modifications as described below.

Optical apparatus

A fibre-optic stub (400 μm core, NA = 0.48, low-autofluorescence fibre; Doric Lenses, MFC_400/430-0.48_2.8mm_ZF1.25_FLT) was stereotaxically implanted in each targeted brain region. A matching fibre-optic patch cord (Doric Lenses, MFP_400/430/1100-0.48_2m_FC-ZF1.25) provided a light path between the animal and a miniature, permanently aligned optical bench, or ‘mini-cube’ (Doric Lenses, FMC5_E1(460-490)_F1(500-540)_E2(555-570)_F2(580-600)_S). One fibre on the ipsilateral side of the viral injection of either 1 μl of AAV2-EF1α-DIO-eNpHR3.0-BFP (Virovek) or 1 μl of AAV5-EF1α-DIO-mCherry (UNC Vector Core) was used to both deliver excitation light to and collect emitted fluorescence from that recording site. The fibre contralateral to the viral injection site was connected to a separate mini-cube (Doric Lenses, FMC6_E1(460-490)_F1(500-540)_E2(555-570)_F2(580-600)_O(628-642)_S) that was attached to a 638 nm laser (Doric Lenses) and was used to deliver excitation light and optogenetic stimulation to and collect emitted fluorescence from that recording site. The far end of the patch cord and each 1.25 mm diameter zirconia optical implant ferrule were cleaned with isopropanol before each recording, then securely attached via a zirconia sleeve.

For the first mini-cube sans laser port, optics allow for the simultaneous monitoring of two spectrally separated fluorophores, with dichroic mirrors and cleanup filters chosen to match the excitation and emission spectra of the voltage sensor and reference fluorophores in use (‘mNeon’ voltage sensor channel: excitation 460–490 nm, emission 500–540 nm; ‘red’ control fluorophore: excitation 555–570 nm, emission 580–600 nm). The mini-cube optics are sealed and permanently aligned and all five ports (sample to animal, two excitation lines and two emission lines) are provided with matched coupling optics and FC connectors to allow for a modular system design.

For the second mini-cube that contains a port for optogenetic stimulation, a 565 nm LED and 555–570 nm filter is used to excite tdTomato, a 580–600 nm filter is used for tdTomato emission, and a 638 nm laser with 628–642 nm filter is used to excite eNpHR. The light used to excite eNpHR does not interfere with the genetically encoded voltage indicator based measurements of synchrony, and the light used to excite tdTomato does not activate eNpHR enough to affect rule-shift performance. This is due to two factors. First, the eNpHR and tdTomato excitation spectra are offset—for example, at 605 nm, eNpHR activation is near its peak whereas relative excitation of tdTomato is <1%. Second, the intensity used to excite tdTomato is just ~0.1 mW, which is ~50-fold less than what was used to activate eNpHR and disrupt rule-shift performance. The mini-cube optics are sealed and permanently aligned and all six ports (sample to animal, three excitation lines and two emission lines) are provided with matched coupling optics and FC connectors to allow for a modular system design.

To perform dual-site voltage indicator recordings, excitation light for each of the two colour channels was provided by a fibre-coupled LED (Center wavelengths 490 nm and 565 nm, Thorlabs M490F3 and M565F3) connected to the mini-cube by a patch cord (200 μm, NA = 0.39; Thorlabs M75L01). Using a smaller diameter for this patch cord than for the patch cord from the cube to the animal is critical to reduce the excitation spot size on the output fibre face and thus avoid cladding autofluorescence. LEDs were controlled by a 4-channel, 10-kHz-bandwidth current source (Thorlabs DC4104). LED current was adjusted to give a final light power at the animal (averaged during modulation, see below) of approximately 200 μW for the mNeon channel (460–490 nm excitation), and 100 μW for the Red channel (555–570 nm excitation).

Each of the two emission ports on the mini-cube was connected to an adjustable-gain photoreceiver (Femto, OE-200-Si-FC; Bandwidth set to 7 kHz, AC-coupled, ‘low’gain of ~5 × 10⁷ V W⁻¹) using a large-core high-NA fibre to maximize throughput (600 μm core, NA = 0.48 (Doric lenses, MFP_600/630/LWMJ-0.48_0.5m_FC-FC).

Note that, for dual-site voltage indicator recordings and optogenetics experiments, two completely independent optical setups were employed, with separate implants, patch cords, mini-cubes, LEDs, a separate laser, photoreceivers, and lock-in amplifiers.

Modulation and lock-in detection

At each recording site, each of the two LEDs was sinusoidally modulated at a distinct carrier frequency to reduce crosstalk due to overlap in fluorophore spectra. The corresponding photoreceiver outputs were then demodulated using lock-in amplification techniques. A single instrument (Stanford Research Systems, SR860) was used to generate the modulation waveform for each LED and to demodulate the photoreceiver output at the carrier frequency. To further reduce crosstalk between recording sites, distinct carrier frequencies (2, 2.5, 3.5 and 4 kHz) were used across sites. Low-pass filters on the lock-in amplifiers were selected to reject noise above the frequencies under study (cascade of 4 Gaussian FIR filters with 84 Hz equivalent noise bandwidth; final attenuation of signals are approximately −1dB (89% of original magnitude) at 20 Hz, −3dB (71% of original magnitude) at 40 Hz, and −6dB (50% of original magnitude) at 60 Hz).

In vivo dual-site voltage indicator imaging

Analogue signals were digitized by a multichannel real-time signal processor (Tucker-Davis Technologies; RX-8). The commercial software Synapse (Tucker-Davis Technologies) running on a PC was used to control the signal processor, write data streams to disk, and to record synchronized video from a generic infrared USB webcam (Ailipu Technology, ELP-USB100W05MT-DL36). Lock-in amplifier outputs were digitized at 3 kHz.

Combined dual-site voltage indicator imaging and optogenetics analysis

Analysis of voltage indicator data was described previously⁸ and was facilitated using the signal processing toolbox and MATLAB (Mathworks), using the following functions: fir1, filtfilt, and regress. All four signals during the entire time series of the experiment (left mNeon, left tdTomato, right mNeon, right tdTomato) were first filtered around a frequency of interest. To quantify zero-phase lag cross-hemispheric synchronization between left and right mNeon signals, a linear regression analysis was performed to predict the right mNeon signal using the following inputs: left mNeon signal, left tdTomato signal, and right tdTomato signal. The goodness of fit is compared to how well the regression works if the left mNeon signal is shuffled, i.e., if a randomly chosen segment of the original left mNeon signal is used, instead of the segment recorded at the same time as the right mNeon signal. R² values are calculated as a function of time using one second segments and compared to the 99th percentile of the distribution of R² values obtained from 100 fits to randomly shuffled data. The fraction of time points at which the R² obtained from actual data exceeds the 99th percentile of the R² values obtained from shuffled data was used to measure zero-phase lag synchronization between the left and right mNeon signals.

This analysis was performed at the time of the decision (for example, immediately following the beginning of digging in one bowl, until the end of digging), and smoothed measurements over a 5-min time window following the time point of interest. The first five trials of the rule shift were analysed. Experiments were performed, scored and analysed blind to virus injected.

Shorter-timeframe resolution dual-site voltage indicator imaging and optogenetics analysis

For high temporal resolution quantification of synchrony between signals from dual-site voltage indicator recordings, we first bandpass-filtered Ace-mNeon and tdTomato signals as described above. Then, we ‘corrected’ the filtered Ace-mNeon signal (to minimize artefacts and noise) by fitting the ipsilateral filtered tdTomato signal via robust linear regression using the robustfit function in Matlab and a time windows of 250 ms. Then, we subtracted off this fit of the tdTomato signal from the filtered Ace-mNeon signal to obtain a corrected Ace-mNeon signal. We then calculated zero-lag cross-correlation between the corrected Ace-mNeon signals from the left and right mPFC across the whole session using 1-s windows.

Combined calcium imaging and optogenetics

Imaging data were collected using a miniaturized one-photon microscope (nVoke2; Inscopix). GCaMP7f signals (calcium activity) were detected using 435–460 nm excitation LED (0.1–0.2 mW), and optogenetic stimulation of eNpHR-expressing axons was performed using a second 590–650 nm excitation LED (1–2 mW light power). nVoke2 software (Inscopix) was used to control the microscope and collect imaging data. Images were acquired at 20 frames per second, spatially downsampled (4×), and were stored for offline data processing. An input TTL from a separate ANY-maze computer (Stoelting Europe) to the nVoke2 acquisition software were used to synchronize calcium imaging and mouse behaviour movies.

Combined calcium-imaging and optogenetics analysis

Calcium-imaging movies were preprocessed using Inscopix Data Processing Software (IDPS; Inscopix). The video frames were spatially filtered (bandpass) with cut-offs set to 0.008 pixel⁻¹ (low) and 0.3 pixel⁻¹ (high) followed by frame-by-frame motion correction for removing movement artefacts associated with respiration and head-unrestrained behaviour. The mean image over the imaging session was computed, and the dF/F was computed using this mean image. The resultant preprocessed movies were then exported into MATLAB, and cell segmentation was performed using an open-source calcium-imaging software (CIAPKG)³⁰. Specifically, a principal component analysis/independent component analysis (PCA/ICA) approach was used to detect and extract regions of interest (presumed neurons) per field of view³¹. For each movie, the extracted output neurons were then manually sorted to remove overlapping neurons, neurons with low signal-to-noise ratio, and neurons with aberrant shapes. Accepted neurons and their calcium activity traces were exported to MATLAB for further analysis using custom scripts as previously described³². In brief, the s.d. (σ) of the calcium movie was calculated and this was used to perform threshold-based event detection on the traces by first detecting increases in dF/F exceeding 2 (over 1 s). Subsequently, events were detected that exceeded 10 for over 2 s and had a total area under the curve higher than 150σ. The peak of the event was estimated as the local maximum of the entire event. For an extracted output neuron, active frames were marked as the period from the beginning of an event until the calcium signal decreased 30% from the peak of the event (up to a maximum of 2 s).

We calculated the similarity of population activity vectors using the ‘cosine similarity’, which is equivalent to computing the normalized dot product between the two vectors, that is:

where x_i and y_i represent the average activity of the ith neuron in the two population activity vectors. Each vector was the average of activity during the 10 s immediately following a choice. We computed the similarity between each pair of vectors, then averaged this similarity across all the pairs from one mouse.

The first five trials of the rule shift were analysed. For Extended Data Fig. 10t–u, the last five initial association trials and the additional initial association trials during optogenetic inhibition were analysed.

Histology and imaging

All mice used for behavioural and imaging experiments were anaesthetized with Euthasol and transcardially perfused with 30 ml of ice-cold 0.01 M PBS followed by 30 ml of ice-cold 4% paraformaldehyde in PBS. Brains were extracted and stored in 4% paraformaldehyde for 24 h at 4 °C before being stored in PBS. Slices 70–100 μm thick were obtained on a Leica VT100S and mounted on slides. All imaging was performed on an Olympus MVX10, Nikon Eclipse 90i, Zeiss LSM510, Zeiss Axioskop2, Zeiss ApoTome.2, and Keyence BZ-X All-in-One Fluorescence Microscope. All mice were verified to have virus-driven expression and optical fibres located in the mPFC. For mice used in parvalbumin immunohistochemistry, 60-μm slices from PV-cre mice injected with AAV2-EF1α-DIO-ChR2-eYFP unilaterally in PFC were obtained on a Leica VT100S and were rinsed twice at room temperature (10 min each) in PBS and incubated overnight at 4 °C with 0.3% Triton X-1000, 0.1% normal donkey serum (NDS) and monoclonal anti-PV antibody (1:1,000; Sigma). Slices were then rinsed twice in PBS (10 min each) at room temperature and incubated with Alexa 688 goat anti-mouse antibody (1:500; Invitrogen) for 3 h at room temperature. Slices were then rinsed twice in PBS (10 min each) at room temperature and coverslipped in mounting medium. Immunofluorescence was then observed with Zeiss ApoTome.2 and images were acquired.

Data analyses and statistics

Statistical analyses were performed using Prism 8 (GraphPad) and detailed in the corresponding figure legends. Quantitative data are expressed as the mean and error bars represent the s.e.m. Group comparisons were made using two-way ANOVA followed by Bonferroni post hoc tests to control for multiple comparisons unless otherwise noted. Paired and unpaired two-tailed Student’s t-tests were used to make single-variable comparisons. Similarity of variance between groups was confirmed by the F test. Measurements were taken from distinct samples and from samples that were measured repeatedly. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Comparisons with no asterisk or ‘NS’ had P > 0.05 and were considered not significant. No statistical methods were used to pre-determine sample sizes but our sample size choice was based on previous studies^6,8 and are consistent with those generally employed in the field. Data distribution was assumed to be normal, but this was not formally tested.

For Fig. 3d,e,k,l, full statistics are: optogenetic inhibition of callosal mPFC PV terminals impairs rule shifts in DIO-eNpHR mice (n = 5) compared to DIO-mCherry controls (n = 4) on days 2 and 3 (two-way ANOVA (task day × virus); interaction: P = 0.0009). d, DIO-mCherry performance did not change (day 1 to 2: P = 0.28; day 1 to 3: P = 0.094; day 2 to 3: P > 0.99). d,e,k,l, Two-way ANOVA (task day × virus) comparing rule-shift (RS) performance across groups (interaction: P < 0.0001), showed no group difference on day 1 but significant impairment on days 2 and 3 for DIO-eNpHR compared to DIO-mCherry and Syn-tdTomato. e, Optogenetic inhibition of mPFC callosal PV terminals impairs rule shifts in DIO-eNpHR mice compared to DIO-mCherry controls (day 1 to 2: P = 0.014; day 1 to 3: P = 0.012; day 2 to 3: P = 0.075). For Fig. 3f,g, full statistics are: 30–50 Hz synchronization is higher after RS errors than after RS correct decisions across task days in controls (n = 4 mice; two-way ANOVA; main effect of trial type: P = 0.0056; day 1: P = 0.004; day 2: P = 0.005; day 3: P = 0.022). g, Gamma synchrony is higher after RS errors than after RS correct decisions for DIO-eNpHR mice on day 1 (no light), but not days 2 and 3 (n = 5 mice; two-way ANOVA (trial type × task day); interaction: P = 0.0039; day 1: P = 0.0056; day 2: P = 0.16; day 3: P = 0.23). Differences in gamma synchrony between RS errors and correct trials are also significantly lower in DIO-eNpHR mice compared to controls on days 2 (two-way ANOVA (trial type × virus); interaction: P = 0.018) and 3 (two-way ANOVA (trial type × virus); interaction: P = 0.034). We then performed two-way ANOVA (task day × virus, type of error) comparing gamma synchrony across groups with appropriate post hoc tests corrected for multiple comparisons; gamma synchrony is lower for DIO-eNpHR mice compared to matched controls on error (interaction: P = 0.049), but not correct trials (interaction: P = 0.93) on both days 2 (P = 0.0089) and 3 (P = 0.016). For Fig. 3k–n, full statistics are: inhibiting callosal terminals does not affect rule shifts in Syn-eNpHR mice (n = 5) compared to controls (n = 4) across days (two-way ANOVA (task day × virus); P = 0.37). k, Performance of controls did not change across days (day 1 to 2: P = 0.48; day 1 to 3: P > 0.99; day 2 to 3: P > 0.99). l, Performance of Syn-eNpHR mice did not change across days (day 1 to 2: P > 0.99; day 1 to 3: P = 0.96; day 2 to 3: P = 0.85). m, Interhemispheric 30–50 Hz synchronization is higher after RS errors than RS correct decisions across days in controls (n = 4 mice; two-way ANOVA; main effect of trial type: P = 0.020; day 1: P = 0.015; day 2: P = 0.049; day 3: P = 0.025). n, Gamma synchrony is higher after RS errors than RS correct decisions for Syn-eNpHR mice on day 1 (no light). This was abolished with light on day 2, then restored in the absence of light on day 3 (n = 5 mice; two-way ANOVA; (trial type × task day); interaction: P = 0.011; day 1: P = 0.037; day 2: P = 0.46; day 3: P = 0.021). Gamma synchrony is lower for Syn-eNpHR mice compared to matched controls on error (two-way ANOVA; interaction: P = 0.045) but not correct trials (interaction: P = 0.74) on day 2 (P = 0.043), but not on day 3 (P = 0.58). Two-way ANOVA followed by Bonferroni post hoc comparisons was used.

For Fig. 4d–f, full statistics are: optogenetic inhibition of callosal PV terminals impairs rule shifts in DIO-eNpHR mice (n = 6) compared to controls (n = 6) and Syn-eNpHR mice (n = 6) on days 2 and 3 (two-way ANOVA (task day × virus); interaction: P < 0.0001). d, Performance of controls did not change (day 1 to 2: P = 0.057; day 1 to 3: P = 0.12; day 2 to 3: P = 0.67). e, Optogenetic inhibition of callosal PV terminals impairs rule shifts in DIO-eNpHR mice (day 1 to 2: P = 0.020; day 1 to 3: P = 0.010; day 2 to 3: P > 0.99). f, Performance of Syn-eNpHR mice did not change (day 1 to 2: P = 0.58; day 1 to 3: P = 0.82; day 2 to 3: P > 0.99). For Fig. 4h,j, full statistics are: optogenetic inhibition changes the similarity of activity vectors specifically in DIO-eNpHR mice (two-way ANOVA, main effect of task day: P = 0.017; task day × group interaction: P = 0.32). h, For controls (n = 6 mice), there is no change across days in the similarity between activity vectors after RS errors and those after correct decisions (day 1 to 2: P = 0.57; day 1 to 3: P = 0.32; day 2 to 3: P = 0.90). i, In DIO-eNpHR mice (n = 6 mice), there is an increase in the similarity between activity vectors after RS errors and those after correct decisions from day 1 (before optogenetic inhibition) to days 2 and 3 (during and after inhibition, respectively) (day 1 to 2: P = 0.044; day 1 to 3: P = 0.0067; day 2 to 3: P = 0.71). j, For Syn-eNpHR mice (n = 6 mice), there is no change across days in the similarity between activity vectors after RS errors and those after correct decisions (day 1 to 2: P = 0.99; day 1 to 3: P = 0.93; day 2 to 3: P = 0.98). Two-way ANOVA to compare the change in activity vector similarity from day 1 to 2 in the DIO-eNpHR group to a group consisting of the eNpHR-negative and synapsin-eNpHR mice, using mouse, day, and day × group interaction as factors revealed a significant day × group interaction (P = 0.040). k–m, The similarity between activity vectors after decisions on error trials during the initial association (IA) and those during the RS. There is increased similarity across days in DIO-eNpHR mice (n = 6; two-way ANOVA (task day × virus); interaction: P = 0.025). k, There is no change in the similarity of population activity vectors after IA errors and RS errors across days in controls (n = 6; day 1 to 2: P = 0.99; day 1 to 3: P = 0.52; day 2 to 3: P = 0.50). l, During and following optogenetic inhibition on days 2 and 3 in DIO-eNpHR mice, there is an increase in the similarity of population activity vectors following IA versus RS error trials (day 1 to 2: P = 0.0038; day 1 to 3: P = 0.0045; day 2 to 3: P = 0.95). m, In Syn-eNpHR mice, there is no change in the similarity of population activity vectors following IA versus RS errors trials across days (n = 6; day 1 to 2: P = 0.41; day 1 to 3: P = 0.90; day 2 to 3: P = 0.24). n, Controls have increases in average activity (fraction of frames in which a neuron is active, averaged across all neurons) during the 10 s following RS errors compared to the 10 s following RS correct decisions on all days (n = 6; two-way ANOVA, main effect of trial type: P = 0.010; day 1: Bonferroni P = 0.022; day 2: Bonferroni P = 0.015; day 3: Bonferroni P = 0.016). o, DIO-eNpHR mice have an increase in average activity after errors that depends on the day (n = 6; two-way ANOVA (task day × trial type); interaction: P = 0.0003). This difference occurs on day 1 (Bonferroni P < 0.0001), but is abolished with light delivery on day 2 (Bonferroni P > 0.99), and continues to be absent even without further light delivery on day 3 (Bonferroni P > 0.99). p, In Syn-eNpHR mice, there is an overall increase in average activity following error trials (n = 6; two-way ANOVA, main effect of trial type: P = 0.0088). This occurs on days 1 (Bonferroni P = 0.038) and 3 (Bonferroni P = 0.016), but not with light delivery on day 2 (Bonferroni P > 0.99). Two-way ANOVA followed by Tukey post hoc comparisons was used unless otherwise noted.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.