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Insula to mPFC reciprocal connectivity differentially underlies novel taste neophobic response and learning in mice

  1. Haneen Kayyal
  2. Sailendrakumar Kolatt Chandran
  3. Adonis Yiannakas
  4. Nathaniel Gould
  5. Mohammad Khamaisy
  6. Kobi Rosenblum  Is a corresponding author
  1. Sagol Department of Neuroscience, University of Haifa, Israel
  2. Center for Gene Manipulation in the Brain, University of Haifa, Israel
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Cite this article as: eLife 2021;10:e66686 doi: 10.7554/eLife.66686

Abstract

To survive in an ever-changing environment, animals must detect and learn salient information. The anterior insular cortex (aIC) and medial prefrontal cortex (mPFC) are heavily implicated in salience and novelty processing, and specifically, the processing of taste sensory information. Here, we examined the role of aIC-mPFC reciprocal connectivity in novel taste neophobia and memory formation, in mice. Using pERK and neuronal intrinsic properties as markers for neuronal activation, and retrograde AAV (rAAV) constructs for connectivity, we demonstrate a correlation between aIC-mPFC activity and novel taste experience. Furthermore, by expressing inhibitory chemogenetic receptors in these projections, we show that aIC-to-mPFC activity is necessary for both taste neophobia and its attenuation. However, activity within mPFC-to-aIC projections is essential only for the neophobic reaction but not for the learning process. These results provide an insight into the cortical circuitry needed to detect, react to- and learn salient stimuli, a process critically involved in psychiatric disorders.

Introduction

Determining salient stimuli from the continuously perceived sensory input is a key component for effective learning that has been shaped throughout evolution. The term ‘salient’ is used in psychology and neuroscience to denote a particularly distinct or important stimulus, or an aspect of it (Yantis and Hillstrom, 1994). Attributing salience to a given cue or stimulus, can be affected by genetics, previous experiences, current psychological and motivational state, stress response, goals and motivation (Puglisi-Allegra and Ventura, 2012; Goldberg et al., 2006). Numerous studies have identified cortical and subcortical brain structures that are involved in the detection of salient stimuli, with the insular cortex (IC) being a key node of the salience network (Gogolla, 2017; Livneh et al., 2017; Uddin, 2015). In particular, the IC is believed to have a critical role in the bottom-up detection of salient events and attention allocation (Menon and Uddin, 2010). Thus, when a salient stimulus is detected, the insula assists in targeting other brain structures that enable access to attention and working memory resources.

Salient experiences are comprised of several dimensions, which include novelty amongst others (Modinos et al., 2015; Schultz, 2013; Winton-Brown et al., 2014). Experiencing a new taste therefore represents a salient experience of novel sensory information, and the memory for this event is dependent on the functionality of the IC (Gal-Ben-Ari and Rosenblum, 2011; Yiannakas and Rosenblum, 2017). Taste recognition, upon which novelty and salience detection inversely depend, relies on a combination of instinctive responses (genetic elements) and brain processes involved in recalling past experiences to help animals cautiously avoid poisonous food, while taking advantage of food that is deemed safe. Therefore, when animals encounter a taste that is completely new to them, they approach it with cautiousness, a phenomenon called taste neophobia (Lin et al., 2015). This neophobic response is the first line of defense, and therefore an important and evolutionarily conserved behavior, manifesting in reluctance to consume the novel taste, for fear of potentially adverse outcomes. Following this tentative consumption of the novel taste, and in the absence of any proceeding aversive visceral consequences, a memory for a safe taste is formed (Osorio-Gómez et al., 2018). Thus, the consumption of this taste gradually increases in the following encounters, a phenomenon called attenuation of neophobia (Barot and Bernstein, 2005; Green and Parker, 1975; Morón and Gallo, 2007). This is an example in which a salient, novel stimulus (1) increases attention which affects the preliminary response, and then (2) initiates incidental encoding for a memory of the new experience as to subsequently prime future adaptive behavior (Borsook et al., 2007; Anderson et al., 2006; McGaugh, 2006). Thus, acting when the sensory information is new, and then learning the new stimulus (familiarization), are both crucial but different components in the process of behavior to a salient, novel stimulus.

The process by which a novel taste becomes a familiar one is partially subserved by the IC (Bermudez-Rattoni, 2014), and the gustatory cortex, located within the medial portion of the anterior IC (aIC) (Gehrlach et al., 2020; Shura et al., 2014; Rolls, 2016; Yiannakas and Rosenblum, 2017). The aIC plays a critical role in the formation and retrieval of novel taste memory (Kayyal et al., 2019; Lavi et al., 2018; Yiannakas and Rosenblum, 2017). Inhibition of protein synthesis or lesions to the IC cause impairment in novel taste learning (Rosenblum et al., 1993). Several molecular changes occur in the IC following novel taste learning (Gal-Ben-Ari and Rosenblum, 2011; Gould et al., 2020), including increased phosphorylation-activation of the extracellular regulated kinase (ERK). Phosphorylated ERK (pERK) levels are increased in the IC following novel taste consumption (Berman et al., 1998) and inhibition of MEK, an upstream kinase of ERK in the IC, impairs novel taste learning (Berman et al., 1998).

Although an accumulating body of work has been directed toward understanding the aIC and the molecular mechanisms by which novel taste memory is formed and retrieved (Adaikkan and Rosenblum, 2015; Merhav et al., 2006; Yefet et al., 2006), the cellular and circuit mechanisms are yet to be deciphered.

Additionally to the aIC, the mPFC is another region that plays a prominent component of the novelty network that is engaged following an encounter with a novel taste stimulus (Morici et al., 2015; Takehara-Nishiuchi et al., 2020). Lesions of the mPFC result in impaired learning of novel tasks (Barker et al., 2007; Devito and Eichenbaum, 2011), including taste learning (Gonzalez et al., 2015). The mPFC is reciprocally connected with the IC, however, whether and how their connectivity contributes to novel taste learning has not been comprehensively examined (Gabbott et al., 2003). Hence, we set out to assess the involvement of aIC-mPFC functional connectivity in the neophobic response evoked during novel taste exposure, as well as novel taste learning. To do so, we measured the correlation between the number of activated, pERK+ neurons within the general aIC and mPFC populations, and specifically within aIC-to-mPFC or mPFC-to-aIC projecting neurons. We found that experiencing a novel taste causes a significant increase in reciprocal aIC-mPFC neuronal activity as measured by pERK+ neurons (Jones et al., 1999). From an electrophysiological perspective, we identify a correlation between taste novelty and increased excitability in aIC-to-mPFC projecting neurons. In order to ascertain behavioral causality of the molecular and electrophysiological correlations we identified, we inhibited the activity of each pathway during the reaction to- or the learning of- a novel taste. We found that chemogenic inhibition of the aIC-to-mPFC pathway resulted both in impaired neophobic response, as well as impaired taste learning and memory. In contrast, inhibition of the opposite, reciprocal, mPFC-to-aIC pathway, caused impairment to the neophobic response only, but not to the learning process.

Our results show that activation of the aIC-mPFC circuit is correlative and necessary for both the neophobic response to novel tastants, as well as memory formation, with functionally discrete reciprocity. This provides part of a functional map of the broader salience system that is yet to be described, as well as providing a guideline for setting the existing molecular knowledge to reveal the unexplored circuit framework.

Materials and methods

Animals

Animals used were 8-to 12-week-old wild type (WT) adult (C57BL/6) male mice. Mice were kept in the local animal resource unit at the University of Haifa in an environment that is temperature-controlled and under a 12 hr dark/light cycle. Water and chow pellet were available ad libitum. All experiments and procedures conducted were approved by the University of Haifa Animal Care and Use committee under Ethical license 554/18 and were in accordance with the National Institutes of Health guidelines for ethical treatment of animals.

Surgery and viral injections

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For all surgeries, naive adult male mice were used as detailed. Mice received i.p. injections of norocarp (0.5 mg/kg), 30 min before surgery and 24 hr later, to minimize pain. Animals were anesthetized by i.p. injection of ketamine and Dormitor (0.5 mg/kg) and then placed in a stereotaxic device (a Model 963 Kopf Instruments stereotaxic injection system). After exposing the scalp, and using bregma and lambda as relevant alignment points, holes were drilled (0.4 nm) in both hemispheres. Mice were injected with AAV construct at the IC (A-P: +0.86; D-V-4; M-L: ±3.4) and/or the mPFC (A-P: 2.34; D-V: −2; M-L:±0.3) or the basolateral amygdala (BLA) (A-P: −1.6; D-V-4.8; M-L: ±3.375), as stated in each experiment. All AAV constructs used in this study were obtained from the viral vector facility of the University of Zurich (http://www.vvf.uzh.ch). All mice used in our studies were injected with 0.25 ul/site/hemisphere of said AAV constructs (physical titer: 4.5 x 10E12 vg/ml) as defined in each experiment. Viral constructs were injected at a rate on 0.1 µl/min, while the syringe was kept in the injection site for 5 min prior and 10 min following delivery, to ensure efficient distribution of the virus. The skin was closed using Vetbond glue, and mice were given 4 weeks of recovery to guarantee viral expression. After recovery, mice were split into individual cages for behavioral experimental purposes.

Immunohistochemistry and quantification

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For perfusion, mice that underwent behavioral experimentation were deeply anesthetized using isoflurane. When the mice were completely anesthetized, they were perfused transcardially using 4% paraformaldehyde (PFA) in 0.1M PBS solution (PBS, MFCD00131855, Sigma-Aldrich).

After perfusion, brains were placed in falcon tubes containing 4% formaldehyde solution overnight at 4°C. On the following day, the solution was replaced with 30% sucrose in 0.1 M phosphate buffered saline (PBS) and brains were incubated for another 48 hr. Afterwards, brains were stored at −80°C. Coronal sections (40 mm thickness) were then collected from the IC (Bregma: 1.10 mm to 0.26 mm) or the mPFC (Bregma 1.94 mm to 1.54 mm) utilizing a Leica cryostat CM 1950. Following sectioning, slices were washed using PBS for three times, and then blocked for 1 hr using a 0.3% bovine serum albumin (10775835001, Sigma-Aldrich), 0.3% triton X-100 (MFCD00128254, Sigma-Aldrich) and 10% fetal bovine (MFCD00132239, Sigma-Aldrich) solution in PBS. After washing three times with PBS, slices were incubated at 4°C with primary antibodies against rabbit phospho-ERK (1:200 D13.14.4E Cell Signaling) and mouse NeuN (1:500 MAB377 MERCK) in blocking solution. On the next day, the primary antibody solution was removed, and slices were washed three times with PBS. Then, slices were kept in the dark in blocking solution containing donkey anti-rabbit Alexa Fluor 647 (1:500 ab150075 Invitrogen) and Goat anti-mouse Alexa Fluor 488 (1:500 A11001 ThermoFisher) secondary antibodies, for 1.5 hr. Slices were then washed three times using PBS, and mounted on glass slides and Vectashield mounting solution containing DAPI (H-1200) was added prior to adding coverslips.

Slices were visualized and images were acquired using a vertical light microscope (Olympus CellSens Dimension ) using ×10 or ×20 magnification. pERK data was subsequently quantified and analyzed in terms of the bilateral number of positive cells/slice using two-way ANOVA (Graphpad Prism ), to examine the respective responses in the general population. The number of rAAV+ cells was similarly quantified and analyzed in the above slices. The images of IC and/or mPFC slices were processed with Image-Pro Plus V-7, Media Cybernetics , pERK+ and rAAV+ neurons were quantified manually in the relevant subregions and layers. To assess the recruitment of the IC-to-mPFC or mPFC-to-IC projecting neurons in the pERK population, the number of cells co-localized with rAAV+ cells was normalized to the total rAAV+ population [(rAAV+pERK+/ rAAV+)*100%] in the respective subregions and layers in slices of the IC or the mPFC. Quantification was done using randomly assigned IDs for individual animals, regardless of treatment. Following quantification, we confirmed treatments, and data were analyzed (Graphpad Prism ).

Behavioral experiments

Mice were randomly allocated to experimental groups. Group size range estimation was based on previously published results using similar methods, as well power calculations (https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html).

Immunohistochemical studies

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Mice aged 8–12 weeks were injected with rAAV-hSyn1-chI-mCherry-WPRE-SV40p(A), rAAV construct, at the mPFC (to label IC-to-mPFC projecting neurons) or at the IC (to label mPFC-to-IC projecting neurons). One month later, animals were individually separated and given 5 acclimation days. Next, mice were water deprived for 24 hr, and were given tap water (for novel saccharin group) or saccharin (0.5% dissolved in tap water, for familiar saccharin group) pipettes for 20 min sessions each day for 6 consecutive days. On the 7th day, all animals were presented with 1 ml of saccharin, and perfused 20 min later, for immunohistochemical analysis.

IC-to-mPFC/ mPFC-to-IC circuit inhibition during novel saccharin exposure, or familiar taste retrieval

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In order to target the IC-to-mPFC circuit for inhibition, mice aged 8–12 weeks were bilaterally injected with AAV8_hEF1a-dlox-hM4D(Gi)_mCherry(rev)-dlox-WPRE-hGHp(A), a Cre-dependent AAV construct expressing the inhibitory DREADD receptor in neurons of the IC. Bilateral injection in the mPFC with rAAV-hSyn1-chI-EGFP_2A_iCre-WPRE-SV40p(A), a retrograde-Cre AAV construct, was also conducted, as to target IC-to-mPFC projecting neurons for inhibition. As a control study, another set of mice were injected with AAV8_hEF1a-dlox-mCherry (rev)-dlox-WPRE-hGHp (A) at the aIC, an mCherry construct without DREADD receptor, while the aforementioned retrograde-Cre AAV construct was injected at the mPFC.

To target mPFC-to-IC projecting neurons for inhibition, the same constructs were used to injections in a different set of mice, with the retrograde-Cre AAV construct being injected in the IC, and the Cre-dependent DREADD injected at the mPFC.

Animals were then given 4 weeks of recovery. Subsequently, animals were individually separated and given 5 days for acclimation. Mice were then water deprived for 24 hr, and given 20 min of pipettes containing tap water for 20 min session for 3 days. Next, animals were i.p. injected with CNO (diluted in saline; 0.5 mg/kg; Enzo) or Saline for control (1% body weight) 1 hr prior to a choice test. Choice test of two pipettes was provided to the mice, with one pipette containing 0.5% saccharin (their first exposure to saccharin) and the other containing water. Drinking volumes for each solution were measured and aversion index scores were calculated (aversion index = volume of water consumed/volume of (water + saccharin) consumed%).

During the subsequent 4 days, mice were presented with unreinforced choice tests of saccharin and water, for 20 min session each day, without any intervention. On the 6th day, animals were only given water in pipettes. Toward examining the importance of the circuitry during familiar taste retrieval, mice were injected on the 7th day with CNO or saline, 1 hr prior to an additional choice test.

Circuit inhibition during aversive taste memory retrieval

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In order to test the importance of IC-to-mPFC projecting neurons in aversive taste memory retrieval, animals were injected with AAV8_hEF1a-dlox-hM4D(Gi)_mCherry(rev)-dlox-WPRE-hGHp(A), a Cre-dependent AAV construct expressing the inhibitory DREADD receptor at the IC, and rAAV-hSyn1-chI-EGFP_2A_iCre-WPRE-SV40p(A), a retrograde AAV Cre construct at the mPFC. 4 weeks after the surgery, mice were individually housed and allowed 5 days for acclimation. Mice were then water deprived for 24 hr prior to being allowed access to water pipettes in 20 min sessions for 3 days. On the following day, mice were trained in CTA by exposing them to saccharin for the first time. Forty min after saccharin consumption, mice were i.p. injected with a 2% body weight dose of LiCl (0.14 M), a malaise inducing agent. Mice were water-restricted for the subsequent 2 days. On the 4th day, mice were injected with CNO or saline, 1 hr before saccharin/water choice test. After 20 min of choice test, drinking volumes were measured and aversion index scores were calculated as above.

Circuit inhibition during innately aversive novel taste exposure

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In order to target IC-to-mPFC projecting neurons for inhibition, mice aged 8–12 weeks were bilaterally injected with AAV8_hEF1a-dlox-hM4D(Gi)_mCherry(rev)-dlox-WPRE-hGHp(A), a Cre-dependent AAV construct expressing the inhibitory DREADD receptor in neurons of the IC, and bilateral injection of rAAV-hSyn1-chI-EGFP_2A_iCre-WPRE-SV40p(A), a retrograde-Cre AAV construct, at the mPFC. Animals were individually separated a month after the injection and given 5 days of acclimation. Mice were then water deprived for 24 hr and were given water pipettes for 20 min each day for 3 days. On the test day, mice were injected with CNO or saline one hour prior to a choice test between water and Quinine (0.04%). The mice were presented with the choice test for 20 min and drinking volumes of each solution was measured. Aversion was calculated accordingly.

IC-to-BLA circuit inhibition during expression of neophobia

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In order to target the IC-to-BLA circuit for inhibition, mice aged 8–12 weeks were bilaterally injected with AAV8_hEF1a-dlox-hM4D(Gi)_mCherry(rev)-dlox-WPRE-hGHp(A), a Cre-dependent AAV construct expressing the inhibitory DREADD receptor in neurons of the IC. Bilateral injection in the BLA with rAAV-hSyn1-chI-EGFP_2A_iCre-WPRE-SV40p(A), a retrograde-Cre AAV construct, was also conducted, as to target IC-to-BLA projecting neurons for inhibition. Animals were then given 4 weeks of recovery, prior to being individually housed and given 5 days for acclimation. Then, animals were water deprived for 24 hr, and given 20 min of pipettes containing tap water for 20 min session for 3 consecutive days. Next, animals were i.p. injected with CNO (0.5 mg/kg; Enzo) or Saline for control (1% body weight) 1 hr prior to a choice test. Choice test of two pipettes was provided to the mice, with one pipette containing 0.5% saccharin (their first exposure to saccharin) and the other containing water. Drinking volumes were measured and aversion was calculated accordingly.

During the subsequent 4 days, mice were presented with unreinforced choice tests of saccharin and water, in 20 min sessions each day, without any additional intervention.

IC-to-mPFC/ mPFC-to-IC circuit inhibition during an open field test

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Mice aged 8–12 weeks were bilaterally injected with AAV construct expressing the inhibitory DREADD receptor in IC-to-mPFC or mPFC-to-IC projecting neurons as previously described. As a control study, another set of mice were injected with a control Cre-dependent mCherry-expressing AAV vector at the aIC and a retrograde-Cre AAV construct at the mPFC. All three groups were i.p. injected with CNO, and 1 hr later, they were placed in a 50×50 cm open-field arena (Noldus Information Technology), for 10 min.

Electrophysiology

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To label neurons in the IC that project to the mPFC, mice were injected with rAAV-hSyn1-chI-mCherry-WPRE-SV40p(A) construct, at the mPFC. One month after the surgery, mice were split to individual cages, and given 5 days of acclimation period. Mice were water deprived for 24 hr, and then presented with water (for novel saccharin group) or saccharin (for familiar saccharin group) for six consecutive days. On the 7th day, mice were presented with 1 ml of saccharin and sacrificed 1 hr later. A cage control group, injected with the rAAV-hSyn1-chI-mCherry-WPRE-SV40p(A) construct at the mPFC, were also sacrificed without any separation nor water restriction procedure. To obtain brain slices containing the aIC, mice were deeply anesthetized with 5%isoflurane and transcardially perfused with 40 ml of ice-cold oxygenated cutting solution containing the following (in mM): 25 NaHCO3, 105 Choline-Chloride, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 25 D-glucose, 1 Na-Ascorbate and 3 Na-Pyruvate. All reagents were commercially obtained from Sigma-Aldrich Israel, except where stated. The 300-µm-thick coronal brain slices were cut with a Campden-1000 Vibrotome using the same cutting solution. The slices were allowed to recover for 30 min at 37°C in artificial CSF (ACSF) containing the following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 D-glucose, 2 CaCl2, and 1 MgCl2, followed by additional recovery for at least 30 min in ACSF at room temperature until electrophysiological recording. The solutions were constantly gassed with carbogen (95% O25% CO2).

Intracellular whole-cell recording

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After the recovery period, slices were placed in the recording chamber and maintained at 32–34°C with continuous perfusion of carbogenated ACSF (2 ml/min). Brain slices containing the aIC were illuminated with infrared light and pyramidal cells were visualized under a differential interference contrast microscope with 10X or 40X water-immersion objectives mounted on a fixed-stage microscope (BX51-WI; Olympus). The image was displayed on a video monitor using a charge-coupled device (CCD) camera (Dage MTI). aIC-to-mPFC projecting neurons were identified by mCherry, and whole cell recordings from aIC to mPFC-projecting neurons were performed using an Axopatch 200B amplifier and digitized by Digidata 1440 (Molecular Devices). The recording electrode was pulled from a borosilicate glass pipette (3–5 MΩ) using an electrode puller (P-1000; Sutter Instruments) and filled with a K-gluconate-based internal solution (in mM): 130 K-gluconate, 5 KCl, 10 HEPES, 2.5 MgCl2, 0.6 EGTA, 4 Mg-ATP, 0.4 Na3GTP and 10 phosphocreatine (Na salt). The osmolarity was 290 mOsm, and pH was 7.3. The recording glass pipettes were patched onto the soma region of rAAV+ pyramidal neurons.

The recordings were made from the soma of aIC pyramidal cells, particularly from layer 2/3 and Layer 5/6. Liquid junction potential (10 mV) was not corrected online. All current clamp recordings were low-pass filtered at 10 kHz and sampled at 50 kHz. Series resistance was compensated and only series resistance <20 MΩ was included in the dataset. Pipette capacitance was ~ 80% compensated. The method for measuring active intrinsic properties was based on a modified version of previous protocols (Kaphzan et al., 2013; Sharma et al., 2018).

Recording parameters

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Resting membrane potential (RMP) was measured 10 s immediately after the beginning of whole cell recording (rupture of the membrane under the recording pipette). The dependence of firing rate on the injected current was obtained by injection of current steps (of 500 ms duration from +50 to 400 pA in 50 pA increments). Input resistance (Rin) was calculated from the voltage response to a hyperpolarizing current pulse (−150 pA). Sag ratio was calculated from voltage response −150 pA. The sag ratio during the hyperpolarizing steps calculated as [(1-ΔVSS/ ΔV max) x 100%] as previously reported (Song et al., 2015). Membrane time constant was determined using a single exponential fit first 100 ms of raising phase of cell response to 1 s −150 pA hyperpolarization step.

For measurements of a single action potential (AP), after initial assessment of the current required to induce an AP at 15 ms from the start of the current injection with large steps (50 pA), a series of brief depolarizing currents were injected for 10 ms in steps of 10 pA increments. The first AP that appeared on the 5 ms time point was analyzed. A curve of dV/dt was created for that trace and the 30 V/s point in the rising slope of the AP was considered as threshold (Sharma et al., 2018). AP amplitude was measured from the equipotential point of the threshold to the spike peak, whereas AP duration was measured at the point of half-amplitude of the spike. The medium after-hyperpolarization (mAHP) was measured using prolonged (3 s), high-amplitude (3 nA) somatic current injections to initiate time-locked AP trains of 50 Hz frequency and duration (10–50 Hz, 1 or 3 s) in pyramidal cells. These AP trains generated prolonged (20 s) AHP, the amplitudes and integrals of which increased with the number of APs in the spike train. AHP was measured from the equipotential point of the threshold to the anti-peak of the same spike (Gulledge et al., 2013). Series resistance, Rin, and membrane capacitance were monitored during the entire experiment. Changes 30% in these parameters were criteria for exclusion of data.

Results

Novel taste experience increases the number of pERK+ cells in mPFC-projecting neurons from inner layers of the aIC

Activity in the IC is correlated and necessary for the formation of taste memory traces (Gal-Ben-Ari and Rosenblum, 2011). The IC displays high connectivity with other brain regions, and these reciprocal interactions facilitate integration of sensory information, valence and reward (Yiannakas and Rosenblum, 2017). Previously, we identified a correlation between an aversive taste valence and activation of aIC-to-BLA projecting neurons (Lavi et al., 2018). We also proved that activity of these projections is necessary for the acquisition and retrieval of an aversive taste memory, but not for attenuating a neophobic response to a novel, non-aversive taste (Kayyal et al., 2019). Given that mPFC activity is crucial for novel taste memory formation (Gonzalez et al., 2015; Uematsu et al., 2015; Mickley et al., 2007) and is correlated with learning novel experiences (Euston et al., 2012), we examined the role of aIC-mPFC reciprocal projections in novel taste learning. Toward that end, WT mice were injected with a retrograde virus containing an mCherry construct in the mPFC, labeling neurons in the IC that project to the mPFC (Figure 1i). As ERK activation is necessary for taste learning (Elkobi et al., 2008), we used pERK, as an indicator of activated neurons (Sweatt, 2001; Adaikkan and Rosenblum, 2012). We quantified the number of pERK+ neurons in 24 aIC slices (bregma 1.10 mm- 0.26 mm) of these mice following novel (one exposure, n=3) or familiar (eight exposures, n=5) taste experiences involving the same taste, saccharin (see Materials and methods, Figure 1a,h). Quantification of pERK+ cells across the aIC demonstrated novel saccharin consumption to be associated with an increased number of activated, pERK+ neurons compared to familiar saccharin, in agreement with the literature (Berman et al., 1998) (unpaired t-test: p=0.0002, t=3.883, DF=114; Figure 1b). The aIC itself is comprised of three major subregions: the agranular (AIC), dysgranular (DIC), and granular IC (GIC) with the 4th cortical layer persisting only in the dorsal portion of the GIC (Figure 7). Within these subregions, experiencing novel saccharin increased the number of pERK+ neurons in the AIC (p=0.04, t=2.094, DF=67; Figure 1c) and the DIC (unpaired t-test: t=3.749, p=0.0003, DF=94; Figure 1d), but not the GIC (unpaired t-test: t=1.340, p=0.184, DF=92; Figure 1e) compared to familiar saccharin. Different cortical layers play different roles in sensory information processing (Dikecligil et al., 2020; Harris and Mrsic-Flogel, 2013). Hence, we examined the expression of pERK in the outer (1,2,3 combined) and inner (5,6 combined) layers. We found that both in outer (unpaired t-test: p=0.0002, t=3.897, DF=70; Figure 1f) and inner layers (unpaired t-test; p=0.002, t=3.121, DF=89; Figure 1g) of the entire aIC, there is a significant increase in pERK+ neurons following novel saccharin experience, compared to the familiar saccharin group. Extensive reciprocal connectivity exists between the IC (mainly the AIC and DIC) and the mPFC (Gabbott et al., 2003), and this may be analogous to the salience network in humans (Uddin, 2015). We therefore hypothesized that aIC-to-mPFC projections have a vital role in novel taste behavior. We quantified pERK+ neurons in the AIC and DIC that co-localized with retrograde-virus labeling, originating from the mPFC. We found a significant increases in the percentage of mPFC-projecting aIC neurons that are pERK+ following consumption of a novel-compared to familiar taste (unpaired t-test: t=3.301, p=0.0015, DF=69; Figure 1j). This effect was evident at the AIC (unpaired t-test: t=3.513, p=0.0008, DF=69; Figure 1k) but not the DIC (unpaired t-test: t=1.353, p=0.181, DF=70; Figure 1l). Looking at the cortical layer distribution, we found that pERK induction in the aIC is observed in both the inner layers (unpaired t-test: t=3.050, p=0.003, DF=92; Figure 1n) and outer layers of the aIC (Mann Whitney test: p=0.0069, U=352; Figure 1m). To test for variability of labeling between novel/familiar saccharin groups, we perfromed an analysis of number of the aIC rAAV+ neurons in its different subregions/layers and found no significant difference in number of rAAV+ neurons between the groups (Figure 1—figure supplement 1a,b).

Figure 1 with 1 supplement see all
Novel taste experience increases number of pERK+ cells in mPFC-projecting aIC neurons.

(a) Schematic representation of novel or familiar taste learning; animals were water restricted in the first day and administered with saccharin (familiar saccharin group) or water (novel saccharin group) in the following 6 days. In the last day, mice were presented with 1 ml of Saccharin 20 min prior to perfusion. Number of pERK+ neurons was quantified in the different subregions and layers of the IC. (b) Number of pERK+ neurons was significantly increased in the total IC following novel (137.4±5.876) compared to familiar (112.9±3.308) saccharin consumption. (c) Number of pERK+ neurons was significantly increased in the AIC following novel (76.6±8.201) compared to familiar (60.62±3.476) saccharin consumption. (d) Number of pERK+ neurons was significantly increased in the DIC following novel (44±1.996) compared to a familiar (35.13±1.270) saccharin consumption. (e) Number of pERK+ neurons was unchanged in the GIC following novel (18.5±1.096) and familiar (16.79±0.6797) saccharin consumption. (f) Number of pERK+ neurons was significantly increased in the outer layers of the IC following novel (76.58±4.103) compared to familiar (60.75±2.016) saccharin consumption. (g) Number of pERK+ neurons was significantly increased in the inner layers of the IC following novel (63.87±3.009) compared to familiar (52.76±1.868) saccharin consumption. (h) Representative coronal IC sections immunostained for pERK (green) and DAPI (blue) from mice injected with retroAAV at the mPFC (red) following novel (upper) and familiar (lower) saccharin. Scale bar, 50 µm, 20x. (i) Stereotaxic injection of rAAV-mCherry construct (red) at the mPFC and its subsequent labeling in the IC. Representative schematic overlays of the Cre-dependent expression of the chemogenetic receptors using the rAAV systems is shown, demonstrating the expressionto be restricted in the aIC and mPFC. Number of double-labeled (pERK+, rAAV+) neurons was calculated as a percentage of all rAAV+ neurons. (j) Percentage of double-labeled neurons of the IC was significantly higher following novel (9.517±1.337) compared to familiar (6.065 ± 0.313%) saccharin consumption. (k) Percentage of double-labeled neurons of the AIC was significantly higher following novel (11.36 ± 2.050%) compared to familiar (6.036±0.303) saccharin consumption. (l) Percentage of double-labeled neurons of the DIC was similar following novel (8.126 ± 2%) and familiar (5.956 ± 0.546%) saccharin consumption. (m) Percentage of double-labeled neurons of the outer layers of the IC was similar following novel (10.08 ± 2.079%) and familiar (5.02 ± 0.381%) saccharin consumption. (n) Percentage of double-labeled neurons of the inner layers of the IC was similar following novel (10.05 ± 1.089%) and familiar (6.562 ± 0.411%) saccharin consumption. Data are shown as mean ± SEM. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.

Figure 1—source data 1

Novel taste experience increases number of pERK cells in mPFC-projecting aIC neurons.

https://cdn.elifesciences.org/articles/66686/elife-66686-fig1-data1-v2.xlsx

Novel taste experience increases excitability in mPFC-projecting neurons of the inner layers of the aIC

Following the correlation between experiencing a novel taste and increased activation of aIC-mPFC projecting neurons as measured by pERK (Figure 1), considering previous evidence regarding the role of pERK on neuronal intrinsic properties (Cohen-Matsliah et al., 2007), we tested the hypothesis that following the experience of a novel taste, aIC-to-mPFC projecting neurons will become more excitable. Toward that end, we once more injected a rAAV mCherry construct in the mPFC (Figure 2b), allowing the labeling of mPFC-projecting neurons of the IC. A month later, different groups of mice were exposed to novel (n=four mice) or familiar saccharin (n=four mice) and were sacrificed one hour later (see materials and methods, Figure 2a) while cage control mice were sacrificed without water restriction regime (n=4). Whole-cell patch-clamp recordings were then performed in slices from aIC-to-mPFC-projecting neurons (Figure 2b). In accordance with our hypothesis, novel taste consumption increased the excitability of mPFC-projections in the inner layers of the aIC (Figure 2c–d), but not the outer layers (Figure 2—figure supplement 1a–j). Intrinsic property changes included RMP (Figure 2—figure supplement 2c) and AP half -width (Figure 2—figure supplement 2e), with depolarized Rin being observed following novel and familiar saccharin consumption, while AP half –width was significantly reduced following novel saccharin consumption compared to the cage controls. In addition, Rin was increased following novel experience compared to the familiar saccharin consumption (Figure 2—figure supplement 2h). Other intrinsic properties, including rheobase, mAHP, firing threshold, time constant and AP amplitude did not show any differences between the groups (Figure 2—figure supplement 2a–j). Interestingly, mPFC-projecting neurons of the outer layers of the aIC did not exhibit any significant differences in their intrinsic properties when similarly comparing novel (n=three mice) and familiar (n=four mice) tastants (Figure 2—figure supplement 2a–j and Table 1).

Figure 2 with 2 supplements see all
Novel taste exposure increases excitability of mPFC-projecting neurons of the inner insular layers.

(a) Schematic representation of novel or familiar taste learning. Animals were water restricted in the first day and administered with saccharin (familiar saccharin group) or water (novel saccharin group) in the following 6 days. Mice were given novel or familiar saccharin and sacrificed 1 hr later. Intrinsic properties were measured in the different layers of the aIC. (b) Whole-cell current-clamp recordings of aIC neurons projecting to mPFC (red). (c, d) The dependence of firing rate on current step magnitude is significantly higher in layers V/VI insular neurons projecting to mPFC following novel compared to familiar taste or cage controls (two-way repeated measurements ANOVA, n=13–14 cells per group; p=0.0009; F (2, 37) = 8.465). Error bars represent SEM; *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.

Figure 2—source data 1

Novel taste exposure increases excitability of mPFC projecting neurons of the inner insular layers.

https://cdn.elifesciences.org/articles/66686/elife-66686-fig2-data1-v2.xlsx
Table 1
Summary of intrinsic properties of aIC-to-mPFC projecting neurons following novel or familiar taste experience.
GroupsRMP
(mV)
mAHP
(mV)
Input resistance
(MΩ)
Sag ratio
(%)
Time constants
(ms)
AP thresh
(mV)
AP Amp
(mV)
AP half-width
(ms)
Rheobase
(pA)
Layer 2/3
IC-mPFC (F.Sacc)
−72.26 ± 2.02 (10)−4.223 ± 0.8426 (10)128.9 ± 15.02 (10)6.96 ±1.633
(10)
21.89 ± 2.855 (10)−30.2 ± 1.845 (10)52.61 ± 3.211 (10)0.696 ± 0.04483 (10)118.2 ± 31.24 (10)
Layer 2/3
IC-mPFC (Novel)
−71.65 ± 1.504
(10)
−3.509 ± 0.8405 (10)128.6 ± 9.645 (10)5.631 ±1.298 (10)19.63 ± 2.623 (10)−33.92 ± 1.109 (10)55.06 ± 2.263 (10)0.703 ± 0.03798 (10)119.9 ± 16.23 (10)
Layer 5/6
IC-mPFC (F.Sacc)
−62.18 ± 1.368 (13) *−4.14 ± 0.6927 (13)106.9 ± 11.32 (13) *12.29 ± 1.993 (13)14.27 ± 1.842 (13)−33.08 ± 2.421 (13)56.4 ± 2.135
(13)
0.6931 ± 0.05582 (13)102.7 ± 13.68 (13) *
Layer 5/6
IC-mPFC (Novel)
−67.21 ± 1.215 (14)−5.163 ± 0.916 (14)150.1 ± 12.94 (14)8.349 ± 2.134 (14)18.4 ± 2.049 (14)−35 ± 1.499
(14)
50.53 ± 2.242
(14)
0.6136 ± 0.03875 (14)68.86 ± 8.601 (14)
Layer 5/6
IC-mPFC (Cage controls)
−68.37 ± 1.177 (13) ++−3.359 ± 0.9374 (13)128.5 ± 10.54 (13)14.11 ± 2.752 (13)18.74 ± 2.406 (13)−33.17 ± 1.884 (13)53.82 ± 2.298 (13)0.8308 ± 0.05237 (13) $81.62 ± 10.71 (13)
  1. *=p<0.05, aIC-to-mPFC L5/6 novel vs familiar saccharin. ++ = p<0.01 aIC-to-mPFC L5/6 cage control vs familiar saccharin. $=p<0.05 aIC-to-mPFC L5/6 novel saccharin vs cage controls.

Taken together, these results demonstrate that following the salient experience of novel taste consumption, deep layer IC-to-mPFC neurons are activated and display increased excitability, thus identifying part of a circuit involved in this form of learning.

Novel taste experience increases activation of aIC-projecting neurons of the mPFC

The mPFC is involved in novelty behavior and the connectivity between the IC and the mPFC is reciprocal (Gabbott et al., 2003; Gehrlach et al., 2020). Considering the present results, we sought to investigate the role of mPFC inputs to the aIC in novel taste learning. Hence, a separate group of mice were injected with the rAAV mCherry-expressing construct in the aIC, labeling neurons in the mPFC that project to the IC (Figure 3h,i). Mice were exposed to novel (first exposure, n=3, Figure 3a) and familiar (eight exposures, n=3) saccharin. As previously done in the IC, the general population of pERK+ neurons were evaluated in the different mPFC subregions - prelimbic (PrL), infralimbic (InfrL), and cingulate cortex (Cg) from eight slices (Bregma 1.94 mm- 1.54 mm). The whole mPFC (all subregions pooled together) showed an increase in activated, pERK+ neurons following novel taste consumption compared to familiar taste (unpaired t-test: t=5.359, DF=14, p=0.0001; Figure 3b). Analysis of the different layers yielded significant effects both in the outer (t=5.835, DF=14, p<0.0001; Figure 3c) and inner (t=4.512, DF=14, p=0.0005; Figure 3d) layers of the mPFC. The changes in the total mPFC are derived from both the Cg1 (t=3.111, DF=14, p=0.0077; Figure 3e), and the PrL (t=4.306, DF=14, p=0.007; Figure 3f), but not the InfrL (t=0.6712, DF=14, p=0.513; Figure 3g). A significant increase in activated mPFC-to-aIC projecting neurons following novel compared to familiar taste was also identified (t=9.549, DF=14, p<0.0001; Figure 3j). The increase in activated aIC-projecting neurons of the mPFC is derived from both outer (t=5.504, DF=14, p<0.0001; Figure 3k), and inner layers (t=9.923, DF=14, p<0.0001; Figure 3i) of the mPFC. Additionally, all mPFC subregions exhibited increased activation of IC-projecting neurons (Cg, t=4.126, DF=14, p=0.001; PrL, t=9.705, DF=14, p<0.0001; InfrL, t=5.141, DF=14, p=0.0001; Figure 3m–o). Analysis of number of rAAV+ neurons for the different subregions/layers of the mPFC showed no significant difference between novel/familiar saccharin groups (Figure 1—figure supplement 1c,d). These results show that the reciprocal aIC-to-mPFC circuit is activated following novel taste consumption.

Novel taste experience increases number of pERK+ cells in aIC-projecting neurons of the mPFC.

(a) Schematic representation of novel or familiar taste learning; animals were water restricted in the first day and administered with saccharin (familiar saccharin group) or water (novel saccharin group) in the following 6 days. In the last day, mice were presented with 1 ml of Saccharin 20 min prior to perfusion.number of pERK+ neurons was quantified in the different subregions and layers of the mPFC. (b) Number of pERK+ neurons of the mPFC was significantly increased following novel (548.1±36.24) compared to familiar (329±18.89) saccharin consumption. (c) Number of pERK+ neurons of the outer layers of the mPFC was significantly increased following novel (198±16.15) compared to familiar (95.75±6.920) saccharin consumption. (d) Number of pERK+ neurons of the inner layers of the mPFC was significantly increased following novel (349±21.67) in comparison to familiar (233.4±14.040) saccharin consumption. (e) Number of pERK+ neurons in the Cg1 was significantly higher following novel (143±17.71) compared to familiar (86.63±4.988) saccharin consumption. (f) Number of pERK+ neurons in the PrL was significantly higher following novel (314±19.68) in comparison to familiar (203.6±16.44) saccharin consumption. (g) Number of pERK+ neurons in the InfrL was similar following novel (90.23±6.423) and familiar (102.6±17.32) saccharin consumption. (h) Representative coronal mPFC sections immunostained for pERK (Green) and DAPI (blue) from mice injected with rAAV at the IC (Red) following novel (upper) and familiar (lower) saccharin. Scale bar, 50 µm, 20x. (i) Stereotaxic injection of rAAV-mCherry construct (red) at the IC and its subsequent labeling in the mPFC. Representative schematic overlays of the Cre-dependent expression of the chemogenetic receptors using the rAAV systems is shown, demonstrating the expressionto be restricted in the aIC and mPFC. Percentage of double labeled (pERK+,rAAV+) neurons was calculated as a percentage of all rAAV+ neurons. (j) Percentage of double-labeled neurons of the mPFC was significantly increased following novel (10.44 ± 0.434%) compared to familiar (4.819 ± 0.397%) saccharin consumption. (k) Percentage of double-labeled neurons of the outer layers of the mPFC was significantly increased following novel (8.49 ± 0.230%) compared to familiar (4.23 ± 0.739%) saccharin consumption. (l) Percentage of double-labeled neurons of the inner layers of the mPFC was significantly increased following novel (11.81 ± 0.561%) compared to familiar (5.296 ± 0.340%) saccharin consumption. (m) Percentage of double-labeled neurons of the Cg1 was significantly increased following novel (8.543 ± 0.633%) compared to familiar (4.897±0.617) saccharin consumption. (n) Percentage of double-labeled neurons of the Prl was significantly increased following novel (12.23 ± 0.665%) in comparison to familiar (4.893 ± 0.358%) saccharin consumption. (o) Percentage of double-labeled neurons of the InfrL was significantly increased following novel (7.744 ± 0.554%) in comparison to familiar (3.261 ± 0.673%) saccharin consumption. Data are shown as mean ± SEM. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.

Figure 3—source data 1

Novel taste experience increases number of pERK cells in aIC-projecting neurons of the mPFC.

https://cdn.elifesciences.org/articles/66686/elife-66686-fig3-data1-v2.xlsx

Activation of aIC-to-mPFC projecting neurons is necessary for both novel taste neophobic responses and memory formation

In many correlative experimental set-ups, it is difficult to dissociate between the cellular activity needed for expressing the behavior from that which is needed to acquire the information for a learning process. Based on the correlation yielded above (Figures 13), between novel taste consumption and the reciprocal activation of aIC to/from mPFC, we sought to identify whether activation of the aIC-to-mPFC part of the circuit is necessary for novel taste neophobic responses and/or familiarization learning. We therefore injected mice with retrograde-Cre virus in the mPFC, and Cre-dependent inhibitory DREADDs (hM4Di) in the aIC (Materials and methods, Figure 4a,b). Although we used a low concentration of CNO, in order to account for any nonspecific effects of CNO administration (Gogolla, 2017), we injected another group of animals with AAV constructs at the aIC and mPFC, resulting in the expression of mCherry, without DREADD receptors in aIC-to-mPFC projections (see Materials and methods). Animals expressing hM4Di in the aIC-to-mPFC projecting neurons were administered either CNO (n=11) or saline (n=10), 1 hr prior to a choice test of novel saccharin (first exposure) and water (Figure 4c). Choice tests between water and saccharin were then carried out over the next 4 days. We found that inhibition of aIC-to-mPFC projecting neurons not only impairs the neophobic response in the first day (unpaired t-test; p=0.0095, t=2.883, DF=19; Figure 4d), but also causes a measurable increase in the neophobic response on the next day (two-way ANOVA: F(1,175)=7.558, p=0.0075; Figure 4e and Figure 4—figure supplement 1a). In order to control for possible effects of CNO, additional group of mice that were injected with control virus that does not express hM4Di in aIC-to-mPFC projections, were treated with CNO (n=7) 1 hr prior to the same experimental design. Aversion toward the novel taste and its attenuation were not different from the saline treated group (Figure 4—figure supplement 1a), which excludes a possible effect of CNO itself.

Figure 4 with 1 supplement see all
aIC-to-mPFC projecting neurons are necessary for both novel taste neophobic response and learning.

WT male mice were injected with retrograde-Cre AAV at the mPFC (green) and the Cre-dependent DREADDs at the IC (red), resulting in expression of inhibitory hMD4Gi in aIC-to-mPFC neurons. (a) Representative images of retrograde-Cre AAV injection in the mPFC and the consequent labeling in the IC (red). (b) Representative images of neurons targeted for inhibition in the IC: Cre-dependent expression of the chemogenetic receptors (red), retrograde-Cre AAV (green) and DAPI (blue). (c) Schematic representation of behavioral test conducted. Mice injected with IC-to-mPC inhibitory DREADDs received intraperitoneal injection of CNO or Saline 1 hr prior to novel saccharin (first exposure) and water choice test, while aversionwas assessed. In the following days, mice were given a choice test without intervention. (d) CNO-injected mice exhibited significantly lower aversion (51.21 ± 8.255%) levels than do Saline-injected controls (78.36 ± 4.33%) in the first choice day (expression of neophobia). (e) CNO-injected mice were significantly more averse to saccharin (87.45 ± 4.336%, compared to saline (54.57 ± 5.708%)) injected controls in the second day. CNO-injected and saline-injected mice showed similar aversion in the third (CNO: 60.70 ± 8.599%, Saline: 54.93 ± 12.68%) fourth (CNO: 51.13 ± 7.804%, Saline: 27.29 ± 14.34%) and fifth (CNO: 45.77 ± 7.852%, Saline: 29.63 ± 6.974%) day of choice tests. Data are shown as mean ± SEM. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.

Figure 4—source data 1

aIC-to-mPFC projecting neurons are necessary for both novel taste neophobic response and learning.

https://cdn.elifesciences.org/articles/66686/elife-66686-fig4-data1-v2.xlsx

Our results indicate a necessity for the circuit in denoting the taste as novel, although it does not rule out its effect on other facets such as taste recognition, retrieval or aversion.

In order to test recognition and retrieval of a familiar taste, we inhibited the circuit on the seventh day, 1 hr prior to a choice test between saccharin and water, once saccharin neophobia has been attenuated and the taste is no longer novel. We observed no significant difference between the treatment (n=5) and control (n=6) groups (unpaired t-test: t=0.2364, DF=9, p=0.8184; Figure 4—figure supplement 1b,c). In order to test whether inhibiting aIC-to-mPFC projections affects taste aversion retrieval, we trained animals in CTA and inhibited the circuit one hour before the retrieval test (see Materials and methods). We observed no significant difference between animals treated with CNO (n=11) or saline (n=11; unpaired t-test: t=0.4114, DF=20, p=0.6852; Figure 4—figure supplement 1d,e).

Furthermore, we inhibited the aIC-to-mPFC part of the circuit prior to a choice test between innately aversive quinine and water (CNO=7, Saline=7), and found no significant difference in aversion between the two groups (unpaired t-test: t=1.102, DF=12, p=0.2919; Figure 4—figure supplement 1f,g). These results therefore indicate that the aIC-to-mPFC part of the circuit is not necessary for the retrieval of a familiar taste, taste aversion or taste recognition, but is vital for both the initial neophobic response, as well as incidental memory formation.

We have previously shown that activity within aIC-to-BLA projecting neurons is not necessary for attenuation of neophobia (Kayyal et al., 2019). However, since activity within the BLA is necessary for the expression of a neophobic response (Shinohara and Yasoshima, 2019; Lin et al., 2018) and in order to better understand the broader circuit involved in the expression of a neophobic reaction, we sought to inhibit aIC neurons projecting to the BLA during the first encounter with novel saccharin. Mice were injected with retrograde-Cre AAV at the BLA, and Cre-dependent inhibitory DREADDs at the IC (see Materials and methods, Figure 5a–b). A month later, mice received an i.p. injection of CNO (n=8) or saline (n=6) one hour prior to a choice test of novel saccharin and water (Figure 5c). Inhibition of aIC-to-BLA projecting neurons caused a reduction in neophobia (unpaired t-test; p=0.007, t=3.243, DF=12) compared to the control group (Figure 5d). However, the attenuation curve did not differ between the two treatments in accordance with our previous report (two way ANOVA: F(1,43) = 0.07789, p=0.7815; Figure 5e), indicating no change in novelty learning. Together, the results show that aIC-to-mPFC projections are necessary both for learning and expressing taste neophobia, while aIC-to-BLA projections are necessary only for the expression of the neophobic response, but not for its attenuation.

Activity within aIC-to-BLA projecting neurons is necessary for novel taste response but not for its attenuation.

WT male mice were injected with retrograde-Cre AAV at the BLA (green) and Cre-dependent DREADDs at the IC (red), resulting in expression of inhibitory hMD4Gi in aIC-to-BLA projecting neurons. (a) Representative images of retrograde-Cre AAV injection in the BLA (green) and the consequent labeling in the IC. (b) Representative images of IC-to-BLA projecting neurons that are targeted for inhibition using Cre-dependent expression of the chemogenetic receptors (red), retrograde-Cre AAV (green) and DAPI (blue). (c) Schematic representation of behavioral test conducted. Mice injected with IC-to-BLA inhibitory DREADDs received intraperitoneal injection of CNO or Saline 1 hr prior to novel saccharin (first exposure) and water choice test, while aversion was assessed. In the following days, mice were given a choice test without intervention. (d) CNO-injected mice exhibited lower aversion (41.11 ± 9.309%) levels than do Saline-injected controls (79.60 ± 5.604%) in the first choice day (unpaired t-test: p=0.007, t=3.243, DF=12). (e) CNO-injected and saline-injected mice showed similar aversion on the second (CNO: 36.26 ± 11.04%, Saline: 40.0 ± 12.98%), third (CNO: 16.06 ± 5.01%, Saline: 18.25 ± 8.920%), fourth (CNO: 26.13 ± 10.52%, Saline: 18.67 ± 12.48%), and fifth (CNO: 6.50 ± 4.153%, Saline: 0.00 ± 0.00%) day of expression of neophobia. Data are shown as mean ± SEM. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.

Figure 5—source data 1

Activity within aIC-to-BLA projecting neurons is necessary for novel taste response but not for its attenuation.

https://cdn.elifesciences.org/articles/66686/elife-66686-fig5-data1-v2.xlsx

Activity of mPFC-to-aIC projecting neurons is necessary for novel taste neophobic response but not memory formation

To better understand the interplay of information between the mPFC and IC and the role the mPFC plays in novelty behavior, we sought to inhibit the directionally opposite element of the pathway, the mPFC-to-aIC projection. Mice were therefore injected with rAAV-Cre at the aIC, and Cre-dependent inhibitory DREADDs at the mPFC (see materials and methods, Figure 6a–b). We then injected mice with CNO (n=8) or Saline (n=5) one hour prior to a choice test between novel saccharin (first exposure) and water (Figure 6c). Inhibition of the mPFC-to-aIC circuit caused a reduction in aversion index during the first choice test (unpaired t-test: t=2.995, DF=11, p=0.0131; Figure 6d). However, it did not affect attenuation of neophobia in the following days (two way ANOVA: F(1,44) = 1.608, p=0.2114; Figure 6e, Figure 6—figure supplement 1a). In order to test whether this was due to an effect on taste identification, we performed another experiment in which we injected animals with CNO (n=4) or saline (n=5) prior to a choice test, conducted after mice were familiarized with saccharin (six exposures; Figure 6—figure supplement 1b). Inhibition of mPFC-to-aIC projecting neurons did not affect aversion after taste familiarization (Figure 6—figure supplement 1c; unpaired t-test: t=0.3018, DF=7, p=0.7716). This indicates that the activation of mPFC-to-aIC projecting neurons is necessary for the neophobic response to a novel taste, but not for the formation of a memory trace, as is additionally seen in the reciprocal aIC-to-mPFC part of the pathway.

Figure 6 with 2 supplements see all
Activity of mPFC-to-aIC projecting neurons is necessary for novel taste neophobic reaction but not for the learning.

WT male mice were injected with Cre-dependent DREADDs at the mPFC and rAAV-Cre at the IC, resulting in expression of inhibitory hMD4Gi in mPFC-to-aIC neurons. (a) Representative images of retrograde-Cre AAV injection in the IC (red) and the consequent labeling in the mPFC. (b) Representative images of mPFC-to-aIC projecting neurons that are targeted for inhibition: Cre-dependent expression of the chemogenetic receptors (red), retrograde-Cre AAV (green) and DAPI (blue). (c) Schematic representation of behavioral test conducted. Mice injected with mPFC-to-IC inhibitory DREADDs received intraperitoneal injection of CNO or Saline 1 hr prior to novel saccharin (first exposure) and water choice test, while aversion was assessed. In the following days, mice were given a choice test without intervention. (d) CNO-injected mice (47.81 ± 11.58%) exhibited significantly lower aversion levels than do saline-injected controls (92.08 ± 2.111%) in the first choice day (expression of neophobia). (e) CNO-injected and saline-injected mice showed similar aversion on the second (CNO: 54.06 ± 13.71%, Saline: 66.84 ± 11.60%), third (CNO: 34.98 ± 11.44%, Saline: 54.76 ± 5.693%), fourth (CNO: 16.85 ± 5.986%, Saline: 34.54 ± 7.856%), and fifth (CNO: 23.80 ± 8.681%, Saline: 10.74 ± 4.712%) day of attenuation of neophobia. Data are shown as mean ± SEM. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.

Figure 6—source data 1

Activity of mPFC-to-aIC projecting neurons is necessary for novel taste neophobic reaction but not for the learning.

https://cdn.elifesciences.org/articles/66686/elife-66686-fig6-data1-v2.xls

To further investigate whether aIC-mPFC inhibition affects anxiety levels, that may be associated with and/or affecting novel taste experiences, we performed an open-field test (Seibenhener and Wooten, 2015). Groups expressing hM4Di in aIC-to-mPFC (n=8) or mPFC-to-aIC projecting neurons (n=9), as well as their respective control virus injected groups (n=8), were all treated with i.p. injections of CNO. One hour later, mice were placed in an open arena (Figure 6—figure supplement 2a). Time spent in the center of the arena and frequency of crossing the center were analyzed. No significant differences was found in both parameters, indicating no effect on anxiety as a consequence of inhibition of aIC-mPFC reciprocal connectivity (Figure 6—figure supplement 2b,c). Aiming to clarify further whether the circuit manipulations affect the detection of the novel taste or anxiety associated with presentation of this taste, we performed another set of experiments, in which we tested the latency toward a novel saccharin and water presentation. Groups expressing hM4Di in aIC-to-mPFC (n=8) or mPFC-to-aIC projecting neurons (n=9) as well as their respective control virus injected groups (n=9) were all injected i.p with CNO. Mice were then presented with a pipette of novel saccharin and time to approach the pipette was recorded. Following 5 s of consumption, the saccharin pipette was removed and 10 s later a water pipette was presented. This routine of saccharin/water presentation was performed twice, consecutively (Figure 6—figure supplement 2d). No significant difference was found between the groups in the time it took the mice to start drinking, either during the first or second presentation of the novel saccharin or water (Figure 6—figure supplement 2e), further indicating no effect on anxiety levels caused by our intervention.

Discussion

Detecting salient, new information from the environment is a crucial aspect of human and animal behavior. The detection and proper reaction to a novel stimulus, and the ability to form a stable memory of it, are two distinct facets of novelty behavior. The molecular and cellular mechanisms underlying these centrally important processes, which are critical for animal survival, have been the subject of much research (Mishkin and Murray, 1994; Schomaker and Meeter, 2015). As a result, numerous studies have explored the role of distinct brain areas and molecular or cellular processes within them that are crucial for salience and novelty detection and learning (Peters et al., 2016; van Kesteren et al., 2012). Although studies conducted in humans show a correlation between several brain structures and novelty detection, little is yet known of the detailed novelty network and directionality in the brain, resulting from causative research (Li et al., 2017). It is therefore important to understand how functionally relevant, distal brain regions act as a circuit to define salience and encode unfamiliar sensory information within a wider salience system.

In the present study, we causally demonstrate that reciprocal activity between the aIC and mPFC, or the BLA, conveys novelty related gustatory information, in one such novelty circuit. Specifically, we show that general neuronal activation in the aIC, as denoted by pERK (Thiels and Klann, 2001), is increased following novel taste experience. This is in line with previous studies, showing that ERK phosphorylation in the aIC allows the formation of a memory trace of the safe taste, and is correlated with taste novelty (Sweatt, 2001; Gutkind, 1998; Kelleher et al., 2004; Thomas and Huganir, 2004; Yiannakas and Rosenblum, 2017). Importantly, our results indicate pERK is increased specifically in the AIC and the DIC subregions of the aIC, which have previously been implicated in processing chemosensory, somatosensory visceral and limbic functions (Jones et al., 2006; Katz et al., 2001; Yokota et al., 2011), emphasizing their relevance in novel taste learning.

Extensive reciprocal connections exist between the IC and the mPFC, that are thought to be an essential component in salience processing both in humans and rodents (Uddin, 2015). Here too, our results show a correlation between the number of activated, pERK+ neurons within reciprocal aIC-mPFC projections following novel taste experience, suggesting plasticity related changes in aIC-mPFC neurons are initiated following taste learning (Impey et al., 1999; Philips et al., 2013; Purcell et al., 2003; Rosenblum et al., 2002). Interestingly, in the aIC this was particularly apparent in the AIC, while in the mPFC the spread of pERK+ neurons was more generalized, being detected across the PrL, InfrL and Cg. The projections of the aIC to the mPFC mainly originate from the AIC (compared to the DIC or GIC), which could be a possible explanation for the specific subregion-effect.

Recent reports indicate the capability of PrL neurons to form ensemble codes for novel stimulus associations within minutes (Takehara-Nishiuchi et al., 2020). This ability seems to underlie the necessity of the mPFC to rapidly detect and selectively encode novel experiences, although our data suggest a role for all mPFC subregions and layers in novel taste processing and learning. Whether and how mPFC subregions, as well as the emerging local circuitry of the aIC are involved in novel taste learning via cell- and subregion-specific mechanisms, on a molecular and cellular level across different phases of learning, will be the subject of future research.

We found that ERK activation within aIC-to-PFC projections correlated with taste novelty, and given ERK involvement in maintaining long-term memory-relevant excitability changes (Cohen-Matsliah et al., 2007), we assessed the intrinsic properties of these projections subsequent to novel taste consumption. In line with the pERK results, we found that aIC-to-mPFC neurons display distinct electrophysiological properties following novel taste consumption. This is in agreement with previous studies in other modalities, showing that novel stimuli and learning of associative experience differentially affect intrinsic properties of neurons in cortical and subcortical regions (Kaczorowski et al., 2012; Sehgal et al., 2013; Sehgal et al., 2014; Song et al., 2015; Song et al., 2015). The increased excitability of aIC-to-mPFC neurons following novel taste experience was observed only in the deep layer of the aIC. Limbic and gustatory information converge in the deep layers of the entire IC, and the main output source of the IC to other cortical and subcortical regions lies within these layers (Kobayashi, 2011; Krushel and van Der Kooy, 1988). In addition, the percentage of taste-coding neurons is higher in the deep layers of the aIC in comparison with the superficial layers (Dikecligil et al., 2020). Our results further strengthen the involvement of the deep layers of the aIC in processing and transmitting taste-related information to other cortical regions, suggesting that these specific molecular and intrinsic changes of aIC-to-mPFC neurons of the deep layers of the aIC allow the formation of a familiar and safe taste memory trace.

Importantly, even though the correlated increase in pERK and in excitability of aIC-mPFC projections following a novel taste demonstrate the involvement of this circuit in novel taste processing, it does not differentiate between the two components of novel taste behavior: novel taste response, or novel taste learning. We thus evaluated if indeed there is a functional aIC-mPFC circuit, by using chemogenetic inhibition to causally examine the reciprocal role of both components, the aIC and the mPFC, in novel taste expression or learning. We found that inhibition of aIC-to-mPFC projecting neurons during novel taste exposure resulted in impaired neophobic responses and novel taste learning. In contrast, inhibiting mPFC-to-aIC projections impaired the expression of neophobia, but not taste memory formation.

This indicates that the mPFC plays a major role in novelty gating, while the detection of novelty and storage of familiar taste memories are both mediated by the aIC. We show that aIC-to-mPFC projections need to be activated during novel taste exposure in order to form a safe taste memory trace, while aIC activation from the mPFC is necessary in order to evoke appropriate behavioral responses to novelty. We thus causally demonstrate that specific connectivity within the aIC and the mPFC allows the identification, the learning and reaction to novel taste stimuli with the memory being formed in the aIC. However, this does not exclude the involvement of other additional stimuli or other circuits that were not the focus of the current study.

The entire IC is an integrative hub for saliency processing. We recently demonstrated that the reciprocal connectivity with the BLA underlies aversive valence processing (Kayyal et al., 2019; Lavi et al., 2018). The BLA is an essential structure for encoding salient stimuli (Fontanini and Katz, 2009) and reward learning (Fontanini et al., 2009), and its signals to the aIC transmit palatability related information (Piette et al., 2012), while facilitating the acquisition and recall of sensory information related to body states, as well as the processing of valence (Gogolla, 2017; Haley and Maffei, 2018; Tye, 2018). Although we have previously shown that activity within aIC-to-BLA projecting neurons is not necessary for novel taste learning, we did not test if it is necessary for the expression of taste neophobia. Here, we show that activity within aIC-to-BLA projecting neurons is essential for the expression of neophobic responses. This goes along with previous data showing that inactivation of the BLA impairs the neophobic response but does not affect the process of attenuation of neophobia, and therefore learning (Lin et al., 2018; Shinohara and Yasoshima, 2019).

Together, our findings suggest that aIC connectivity to different brain areas differentially conveys the novelty state (aIC-to-mPFC/BLA), while following learning-induced plasticity, a now modified circuit state underlies the encoding of the familiarized taste (aIC-to-mPFC but not to BLA) (Figure 7). A functional reciprocal connectivity exists between the BLA and the mPFC (Yizhar and Klavir, 2018), that is of great importance in relation to drinking behavior and water intake (Zhao et al., 2020). In addition, activity within BLA-mPFC projections is necessary for valence-specific behaviors (Yizhar and Klavir, 2018) including innate fear responses (Jhang et al., 2018). Hence, our results do not exclude the possibility of mPFC-BLA activity during novel taste experience and/or learning, but rather suggest a triage of IC, BLA and mPFC participation in novel taste behavior.

A model for the circuits involved in novel taste expression and learning.

Detecting, responding and remembering salient information is a vital property of animal behavior. We show here, for the first time, that the interplay between the aIC and the mPFC in the mammalian brain grasps within it novelty related data, as part of a salience network. Both aIC-mPFC and aIC-to-BLA reciprocal connections are necessary for exhibiting a neophobic response; however, the aIC-mPFC is necessary also to form a memory for a familiar safe taste. In addition, the acquisition and retrieval of learned associations between novel taste and aversive visceral information requires the activity of aIC-to-BLA projecting neurons (Kayyal et al., 2019; Lavi et al., 2018). Together, it places the insula as an integrator of two facets of taste saliency; the novel/familiar axis and aversive/appetitive axis. In addition to the necessity of aIC-mPFC interaction in taste novelty behavior and learning, a recent study reported a prominent role of mPFC-to-AIC in learning of novel olfactory tasks (Zhu et al., 2020). However, it is yet to be defined if and how the IC-mPFC circuitry encodes saliency for other modalities.

There is a growing body of evidence showing that impairment in entire IC-mPFC circuitry is found in schizophrenia, addiction, and depression (Cisler et al., 2013; Penner et al., 2016; Samara et al., 2018), raising the importance for further investigation of this circuit and its dysfunction in patients. Defining the molecular and cellular mechanisms by which salience of sensory or innate information is processed and defined remains a key question in basic and clinical neuroscience. Our results suggest that while the mPFC plays a critical role in reacting to a novel cue, the aIC is essential both in reacting and in learning of the novel stimulus. This suggests that proposed non-invasive treatments for schizophrenia or addiction (such as transcranial magnetic stimulation therapy) could benefit from regiments that target the IC and/or other salience network components, while the integrity of these networks could be of particular pathognomonic value in the developing and adult brain.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures.

References

Decision letter

  1. Mathieu Wolff
    Reviewing Editor; CNRS, University of Bordeaux, France
  2. Laura L Colgin
    Senior Editor; University of Texas at Austin, United States
  3. Shauna L Parkes
    Reviewer; CNRS, France
  4. Jelena Radulovic
    Reviewer; Northwestern University, United States
  5. Steven A Kushner
    Reviewer; Erasmus MC University Medical Center, Netherlands

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This work is of interest to neuroscientists in the field of learning and memory as well as those interested in sensory processing and cortico-cortical interactions. It defines reciprocal cortical pathways that differentially support taste novelty behaviour and learning. Overall, the conclusions are well-supported by the data.

Decision letter after peer review:

Thank you for submitting your article "Insula to mPFC Reciprocal Connectivity Differentially Underlies Novel Taste Neophobic Response and Learning" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Laura Colgin as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Shauna L Parkes (Reviewer #1); Jelena Radulovic (Reviewer #2); Steven A Kushner (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. Overall, the reviewers found the study interesting and the data provided compelling. The reviewers also pointed the following list of items that you need to address in your revised manuscript. For the revised version, please indicate in the title that the biological model was the mouse in this study (this is an eLife's policy).

Essential revisions:

1. Interpretation of Data

Based on the data provided, is the cortical circuit of interest specific to taste? Can we delineate whether the effective circuit manipulations affect the detection of the novel taste or anxiety associated with presentation of this taste, or both? Providing additional behavioral data (not necessary new experiments but new measures – for example on latency to approach the new taste, number of approaches or total time spent sniffing the bottle – which would indicate novelty detection (or lack thereof) independently of saccharose consumption) or a more thorough discussion of these issues is needed.

2. Include BLA data in the main text

The comparison between IC-to-mPFC versus IC-to-BLA is actually a strength of this manuscript. It supports the specificity of the IC-to-mPFC results and allows us to appreciate the broader circuity at work. Please incorporate the data in the main text (so figures as main figures, not supplements) and discuss them accordingly in the manuscript.

3. Scopolamine data

The scopolamine data were found interesting but with specific issues. The authors could decide to remove them entirely from the manuscript to simplify a take-home at the circuit-level. The authors could also keep the data but they will then need to address some issues:

– Can the authors clarify if increased excitability in mPFC-projecting neurons in the IC truly reflects a learning process?

– Figure 3., an alternative interpretation is that mAchR activation may be required at baseline for maintaining intrinsic properties of aIC neurons (F/I curves for novel vs. familiar compared to saline vs. scopolamine are remarkably similar, the underlying physiological mechanism appears divergent, given the relative discordances in RMP, rheobase, input resistance, and AP half-width [Figures 2/3 and S2/S3]).

– Can we really connect slice data with behavioral, circuit-level data here?

4. Controls

Better CNO controls (especially given differences in size groups) are warranted (controls receiving CNO or in vitro slices).

AAV controls; the selectivity of promoters (cell specificity of infected cells) and cre-dependent constructs (i.e. potential leakage of transgene expression without Cre) need to be examined.

Providing baseline levels for pERK (some areas have elevated baseline activity or could be sensitive to the deprivation protocol) and ideally total numbers of AAV+ cells /layer (in addition to the pErk/AAV %) as this will help estimate the variability of labeling between different animals and the overall number of mPFC-projecting neurons would be much appreciated.

5. Figures

Many figures need to have clearer indication of the layers and areas used for quantification (especially 1h, 4h, sup5b)

Reviewer #1:

Kayyal et al. investigated the role of reciprocal pathways between medial prefrontal cortex and insular cortex in novel taste neophobia and memory formation in mice. By combining both correlational (functional neuroanatomy and electrophysiology) and causal techniques (chemogenetics), the authors show that mPFC-projecting neurons in the IC are required for both novel taste expression (neophobia) and taste learning whereas IC-projecting neurons in the mPFC are only involved in the former process. The authors show that consumption of a novel taste increases (1) the number of pERK+ neurons in mPFC-projecting neurons in IC and IC-projecting neurons in mPFC, and (2) the excitability of mPFC-projecting neurons in IC, which is also shown to be ACh-dependent. Finally, using a dual-virus chemogenetic approach to target projection-defined neurons, the authors show that inhibition of the IC-to-mPFC pathway hinders both the expression of taste neophobia and its attenuation (i.e., learning). By contrast, the mPFC-to-IC pathway attenuates only the expression of neophobia but not its attenuation. Demonstrating a differential involvement of reciprocally connected cortical regions is innovative and is likely to be of interest to a fairly broad audience in the field of learning and memory.

One of the major strengths of this manuscript is its use of several complementary approaches that, together, inspire a high level of confidence in the results. The authors also include several behavioural and anatomical controls. For instance, the specificity of the IC-to-mPFC in appetitive taste learning is further supported by comparing the involvement of this pathway to the IC-to-BLA pathway, which is shown to be required for taste neophobia but not attenuation of neophobia (i.e., appetitive taste learning).

Overall, the conclusions are well-supported by the data but there are some weaknesses. Most importantly, the extent to which these results are generalizable to other salient stimuli remains unclear and the visual representation and control of the viral expression (for cre-dependent DREADD virus and retro-AAV) is insufficient.

Weaknesses

1. The authors have not sufficiently addressed whether the involvement of this cortical circuitry in salience and novelty processing is strictly limited to taste. The extent to which the reciprocal interactions between mPFC and IC are also needed to "detect, react to, and learn about" other (non-taste related) sensory stimuli remains unclear. While it is understandable that the authors focus their discussion on interactions between IC and mPFC, there is also no discussion (or mention) of the possible involvement of an mPFC-BLA pathway in novel taste behaviour, particularly in the initial expression of a neophobic response. This seems important given the results with quinine (an innately aversive yet still novel taste) indicate that the involvement of the mPFC-IC reciprocal pathways in novel taste behaviour is not ubiquitous.

2. There is no demonstration of the cre-dependency of the AAV construct expressing DREADD at this titre. Also, the visual representation/quantification of the virus expression (both injection sites and retrograde labelling) is insufficient. For example, the injection site of the retro-AAV in Figure 1i appears to predominately target PL but the full extent of the injection site is unclear (same comment for Figure 5a, where M2 also appears to have been targeted). In Figure 4, magnified images of mPFC are shown but we are not provided with a ROI on the map image (Figure 4i). It is also impossible to assess the number of IC projecting neurons in the subregions (PL, Cg, IL). The image provided (Figure 4i) does not actually show IL at this anteroposterior level and there appears to be very little labelling in Cg (although it is difficult to see at this magnification).

3. While an explicit CNO control group is lacking in this paper, any concerns regarding the effect of CNO per se on taste behaviour are somewhat alleviated by the demonstration that CNO injection (inhibition of aIC-mPFC pathway) does not impair taste recognition and retrieval of a familiar taste. Indeed, mice injected with CNO (n=11) but not saline (n=10) show an impaired neophobic response on day 1 (unfamiliar taste) and an increase in the neophobic response on the next (CNO-free) day. When tested on day 7 (when the taste is now familiar), CNO has no effect indicating that recognition and retrieval of a familiar taste is intact. However, on day 7, the group sizes have been reduced to CNO (n=6) and control (n=5). Why are these group sizes different if the same animals were tested? And if these are different mice, why?

4. ACh release in IC is increased following consumption of an unfamiliar taste. The authors showed that novel taste consumption increased excitability in mPFC-projecting neurons in the IC and this excitability was reduced in scopolamine injected mice. However, the authors did not test the mPFC-to-IC pathway. I imagine that the authors would not expect increased excitability in this pathway if its role is limited to expression of neophobia and not learning. But, unfortunately, without this complementary experiment, it remains unclear. Following the same logic, it is unknown if this reduction in excitability is specific to blocking ACh receptors, even if one might expect that it would be ACh-dependent (and NMDA-independent for example) if the increased excitability truly reflects a learning process.

Reviewer #2:

While I am in general enthusiastic about this paper, I have several suggestions that I believe might strengthen the manuscript and provide some clarifications regarding the research topic:

1. The key question is related to the overall interpretation that focuses on salience/novelty detection. I found it difficult to delineate whether the effective circuit manipulations affect the detection of the novel taste or anxiety associated with presentation of this taste, or both. I know that these may not be easy to detangle, but I think that consideration of all of these interpretations is needed to fully understand the behavioral effects during expression of neophobia. This can be addressed to some extent by providing additional behavioral data, for example on latency to approach the new taste, number of approaches or total time spent sniffing the bottle, which would indicate novelty detection (or lack thereof) independently of saccharose consumption. Or with a control group receiving a familiar taste (water) in a novel bottle, etc. The authors may already have performed such control experiments, and if so, I would suggest presenting such data to facilitate interpretation. This issue is especially relevant in view of the findings that impaired "expression of neophobia" can nevertheless result in taste memory formation (mPFC to AIC projection results).

2. I would also find it useful to show the baseline levels of pErk (naïve mice) and after water deprivation only in the AIC and mPFC because some areas have elevated baseline activity or could be sensitive to the deprivation protocol. It would also be helpful to see the total numbers of AAV+ cells /layer (in addition to the pErk/AAV %). This will help estimate the variability of labeling between different animals and the overall number of mPFC-projecting neurons.

3. The first figures need to have clear indication of the layers and areas used for quantification.

4. It would be helpful to demonstrate (and validate) the CNO effects in slices.

5. I found two aspects of the work underdeveloped: scopolamine and BLA. I would suggest removing the BLA data from this report because all of it is in the supplement and then suddenly shows in the main Figure denoting the circuit. Strengthening the AIC-mPFC circuit model would suffice in my view. The cholinergic aspect would need much more work to integrate because the slice data are very interesting, yet there are no supporting behavioral or circuit data deepening the understanding of the AIC inner layer cholinergic system in the observed effects. If such link is not easy to establish, I would also tend to omit these data and maybe replace them with the retrieval findings. Alternatively, the discussion on cholinergic mechanisms needs to be much stronger and in tighter connection with the demonstrated circuit.

Reviewer #3:

The Rosenblum lab has conducted an important series of experiments to dissect the causal influence of the aIC-to-mPFC connection in distinct aspects of novel taste learning and its adaptive attenuation with subsequent repeated exposure (familiar taste learning). They have used a combination of retrograde labeling to enable visualization of bidirectional monosynaptic connectivity between IC and mPFC, together with pERK labeling to infer in vivo neuronal activation, ex vivo whole cell electrophysiological recordings, and DREADD manipulations to establish the causality of defined connections at distinct cognitive phases during novel taste learning and the subsequent transition to neophobic attenuation with repeated exposure. Overall, the results make an important contribution to our understanding of the cognitive function and plasticity this important neural circuit.

The conclusions of this paper are mostly well supported by data, but a few aspects could be further clarified:

1. AAV8-EF1a and rAAV-hSyn1 promotors for reciprocal aIC-to-mPFC connectivity studies. The EF1a and hSyn1 promoters drive overlapping expression in a broad diversity of neuronal cell types, which leaves open the possibility that distinct classes projection neuron types may be involved. This is particularly relevant for interpretation of the result of the pERK labeling and DREADD inhibition experiments.

2. An alternative explanation for the results in Figure 2 of the novel vs. familiar electrophysiological comparison is that familiar saccharin exposure might decrease intrinsic excitability. Similarly, an alternative explanation for the scopolamine results in Figure 3 is that mAchR activation is required at baseline for maintaining intrinsic properties of aIC neurons. This is especially relevant because although the F/I curves for novel vs. familiar compared to saline vs. scopolamine are remarkably similar, the underlying physiological mechanism appears divergent, given the relative discordances in RMP, rheobase, input resistance, and AP half-width (Figures 2/3 and S2/S3).

3. Previous reports have documented the potential confound on behavioral studies with the use of CNO. Therefore, it would be helpful to confirm whether potential actions of CNO on endogenous neurobiology – independent of its function as a ligand for the DREADD receptor – may be influencing results of key experiments in the manuscript. In particular, it is notable that administration of CNO is associated with a consistent decrease in expression of neophobia across multiple experimental conditions (Figures 5, 6 and S5).

https://doi.org/10.7554/eLife.66686.sa1

Author response

Essential revisions:

1. Interpretation of Data

Based on the data provided, is the cortical circuit of interest specific to taste? Can we delineate whether the effective circuit manipulations affect the detection of the novel taste or anxiety associated with presentation of this taste, or both? Providing additional behavioral data (not necessary new experiments but new measures – for example on latency to approach the new taste, number of approaches or total time spent sniffing the bottle – which would indicate novelty detection (or lack thereof) independently of saccharose consumption) or a more thorough discussion of these issues is needed.

We thank the reviewers for this constructive comment.

In our study we tested whether the aIC-mPFC inhibition affects novel taste detection by inhibition of the circuit prior to quinine (novel, innately aversive tastant), which did not affect the neophobic reaction, indicating no changes in novel taste detection.

Following the important comment, we tested whether inhibiting aIC-mPFC pathway affects anxiety levels during an open-field test through testing of exploratory behavior (Seibenhener & Wooten, 2015). Inhibiting aIC-mPFC pathway did not affect the time spent in the center of the arena nor the frequency in crossing the center (new Figure 6—figure supplement 2a, b), indicating no effect on anxiety-related behavior.

Furthermore, we tested the latency towards approaching the novel tastant as suggested by the reviewer, following aIC-mPFC inhibition. No difference was found in latency towards the novel taste nor the water pipettes (new Figure 6—figure supplement 2d,e), indicating no effect on novelty detection or anxiety.

Recent work reported an essential role of mPFC-to-aIC interplay in novel olfactory tasks (Zhu et al., 2020). Although we tested the necessity of aIC-mPFC circuitry in novel taste experience, we do not exclude its involvement in encoding of other sensory stimuli as suggested by the reviewer. Hence, we addressed this important issue in the Discussion section.

2. Include BLA data in the main text

The comparison between IC-to-mPFC versus IC-to-BLA is actually a strength of this manuscript. It supports the specificity of the IC-to-mPFC results and allows us to appreciate the broader circuity at work. Please incorporate the data in the main text (so figures as main figures, not supplements) and discuss them accordingly in the manuscript.

We appreciate the reviewer’s comment, which has led us to greatly improve the flow and clarity of the paper. BLA data was incorporated to the main text as suggested by the reviewers.

3. Scopolamine data

The scopolamine data were found interesting but with specific issues. The authors could decide to remove them entirely from the manuscript to simplify a take-home at the circuit-level. The authors could also keep the data but they will then need to address some issues:

– Can the authors clarify if increased excitability in mPFC-projecting neurons in the IC truly reflects a learning process?

– Figure 3., an alternative interpretation is that mAchR activation may be required at baseline for maintaining intrinsic properties of aIC neurons (F/I curves for novel vs. familiar compared to saline vs. scopolamine are remarkably similar, the underlying physiological mechanism appears divergent, given the relative discordances in RMP, rheobase, input resistance, and AP half-width [Figures 2/3 and S2/S3]).

– Can we really connect slice data with behavioral, circuit-level data here?

We thank the reviewer for raising this important issue, and accordingly following debating, we removed the scopolamine data from the manuscript.

Slice data has its known limitations as mentioned by the reviewer. However, previous published slice electrophysiology data (Bloodgood et al., 2018; Campanac et al., 2013; Dunn et al., 2019; Otis et al., 2018; Pignatelli et al., 2019; Sano et al., 2014; Santini et al., 2008; Santini & Porter, 2010; Sehgal et al., 2014; Soler-Cedeño et al., 2016; Song et al., 2015; Viosca et al., 2009; Wayman & Woodward, 2018; Whitaker et al., 2017; Yiannakas et al., 2021; Yiu et al., 2014), showed an important correlation between circuit level changes and increased excitability and intrinsic properties in specific neurons, following learning processes. Importantly, we are aware of the limitations and continued the examination using causative relation in the behaving mice.

4. Controls

Better CNO controls (especially given differences in size groups) are warranted (controls receiving CNO or in vitro slices).

AAV controls; the selectivity of promoters (cell specificity of infected cells) and cre-dependent constructs (i.e. potential leakage of transgene expression without Cre) need to be examined.

Providing baseline levels for pERK (some areas have elevated baseline activity or could be sensitive to the deprivation protocol) and ideally total numbers of AAV+ cells /layer (in addition to the pErk/AAV %) as this will help estimate the variability of labeling between different animals and the overall number of mPFC-projecting neurons would be much appreciated.

We thank the reviewers for highlighting the need of further extension of our control experiments. In response to these important points raised by the reviewers, we performed additional experiments.

In order to address how CNO administration by itself affect classical neophobia or its attenuation, control mice (injected with retro-Cre AAV construct at the mPFC, and AAV8_hEF1a-dlox-mCherry (rev)-dlox-WPRE-hGHp (A), an mCherry construct without DREADD receptor, at the IC.) were prepared, and similar behavioral taste-tests were performed. There was no difference in aversion towards novel tastant (or its attenuation) following CNO administration in control-virus injected mice and the saline injected group (new Figure 4—figure supplement 1a). This result indicates no effect of CNO in taste behavior (in the concentration we used).

Regarding cell-specificity of infected cells: Indeed, different neuronal populations (excitatory and inhibitory) might have been targeted by our viral vector containing the synapsin promoter. However, from the literature (Baker et al., 2018; Gehrlach et al., 2020; Greig et al., 2013; Molyneaux et al., 2007; Parnavelas, 2000), we know that most cortical projections (>95%) are excitatory neurons. In addition, and in parallel, this is also notable in our electrophysiological measurements by which all neurons patched show a pyramidal-neuron-like properties. Hence, the probability of targeting inhibitory neurons is low to nothing.

As for the concern regarding potential leakage of transgene expression without Cre: we have used all cre-dependent virus -double inverted floxed (AAV8_hEF1a-dlox-hM4D(Gi)_mCherry(rev)-dlox-WPRE-hGHp(A), aiming for high specificity to cre recombination and less likely to the leakage. The expression of the target gene is specific in cells that express Cre recombinase protein. (Tervo et al., 2016; Zerbi et al., 2019).

Following the reviewers' comment and in order to further clarify the aIC-mPFC anatomical connectivity, we measured and provided data for total numbers of rAAV+ cells in the different subregions/layers (Figure 1—figure supplementary 1a-d). Number of rAAV+ neurons in the aIC and the mPFC was similar both in novel and familiar saccharin groups, indicating no variability in injections/labeling between the groups.

5. Figures

Many figures need to have clearer indication of the layers and areas used for quantification (especially 1h, 4h, sup5b)

As suggested by the reviewer, we amended the figures to improve clarity. Clearer indication of the areas and layers examined was provided in the figures.

Reviewer #1:

Kayyal et al. investigated the role of reciprocal pathways between medial prefrontal cortex and insular cortex in novel taste neophobia and memory formation in mice. By combining both correlational (functional neuroanatomy and electrophysiology) and causal techniques (chemogenetics), the authors show that mPFC-projecting neurons in the IC are required for both novel taste expression (neophobia) and taste learning whereas IC-projecting neurons in the mPFC are only involved in the former process. The authors show that consumption of a novel taste increases (1) the number of pERK+ neurons in mPFC-projecting neurons in IC and IC-projecting neurons in mPFC, and (2) the excitability of mPFC-projecting neurons in IC, which is also shown to be ACh-dependent. Finally, using a dual-virus chemogenetic approach to target projection-defined neurons, the authors show that inhibition of the IC-to-mPFC pathway hinders both the expression of taste neophobia and its attenuation (i.e., learning). By contrast, the mPFC-to-IC pathway attenuates only the expression of neophobia but not its attenuation. Demonstrating a differential involvement of reciprocally connected cortical regions is innovative and is likely to be of interest to a fairly broad audience in the field of learning and memory.

One of the major strengths of this manuscript is its use of several complementary approaches that, together, inspire a high level of confidence in the results. The authors also include several behavioural and anatomical controls. For instance, the specificity of the IC-to-mPFC in appetitive taste learning is further supported by comparing the involvement of this pathway to the IC-to-BLA pathway, which is shown to be required for taste neophobia but not attenuation of neophobia (i.e., appetitive taste learning).

Overall, the conclusions are well-supported by the data but there are some weaknesses. Most importantly, the extent to which these results are generalizable to other salient stimuli remains unclear and the visual representation and control of the viral expression (for cre-dependent DREADD virus and retro-AAV) is insufficient.

Weaknesses

1. The authors have not sufficiently addressed whether the involvement of this cortical circuitry in salience and novelty processing is strictly limited to taste. The extent to which the reciprocal interactions between mPFC and IC are also needed to "detect, react to, and learn about" other (non-taste related) sensory stimuli remains unclear. While it is understandable that the authors focus their discussion on interactions between IC and mPFC, there is also no discussion (or mention) of the possible involvement of an mPFC-BLA pathway in novel taste behaviour, particularly in the initial expression of a neophobic response. This seems important given the results with quinine (an innately aversive yet still novel taste) indicate that the involvement of the mPFC-IC reciprocal pathways in novel taste behaviour is not ubiquitous.

We sincerely thank the reviewer for their valuable comments and suggestions.

Both the IC and the mPFC are heavily implicated in learning of novel sensory stimuli (Bermudez-Rattoni et al., 2005; Gogolla, 2017; Morici et al., 2015; Wang et al., 2020) and the connectivity between them was found to be crucial in learning novel olfactory tasks (Zhu et al., 2020). This indeed raises the possible involvement of aIC-mPFC circuitry in reacting-to and learning of sensory modalities other than taste learning, which we addressed in the Discussion section as suggested by the reviewer.

The mPFC and the BLA are reciprocally and functionally connected (Yizhar & Klavir, 2018). This interplay has been shown to be crucial for learning of valence-specific behaviors (Yizhar & Klavir, 2018) including water intake (Zhao et al., 2020) and in innate fear responses (Jhang et al., 2018). Indeed, it is reasonable to suggest that the interplay between the BLA and the mPFC plays a role in novelty detection and learning as mentioned by the reviewer, and hence we mentioned it in the discussion.

A more thorough debate of the interplay between the BLA and the mPFC was added to the discussion, as well as the involvement of the aIC-mPFC circuit in other modalities.

2. There is no demonstration of the cre-dependency of the AAV construct expressing DREADD at this titre. Also, the visual representation/quantification of the virus expression (both injection sites and retrograde labelling) is insufficient. For example, the injection site of the retro-AAV in Figure 1i appears to predominately target PL but the full extent of the injection site is unclear (same comment for Figure 5a, where M2 also appears to have been targeted). In Figure 4, magnified images of mPFC are shown but we are not provided with a ROI on the map image (Figure 4i). It is also impossible to assess the number of IC projecting neurons in the subregions (PL, Cg, IL). The image provided (Figure 4i) does not actually show IL at this anteroposterior level and there appears to be very little labelling in Cg (although it is difficult to see at this magnification).

We thank the reviewers for raising this important issue. We agree that electrophysiological demonstration and quantification of the effects of CNO in slices expressing inhibitory DREADDs is fundamental to the credibility of the results outlined in the manuscript. We have previously shown a proof of concept for our viral tools in a study published previously (Kayyal et al., 2019) using the rAAV Cre and the cre-dependent DREADDs in IC-to-BLA projecting neurons.

As recommended by the reviewer, we have included overlays confirming that our injection was confined within the aIC or mPFC (Figure 1 and Figure 3), in inhibitory DREADD as well as in correlative pERK experiments. We believe that the above indeed improves the clarity of the presented images as suggested by the reviewer.

3. While an explicit CNO control group is lacking in this paper, any concerns regarding the effect of CNO per se on taste behaviour are somewhat alleviated by the demonstration that CNO injection (inhibition of aIC-mPFC pathway) does not impair taste recognition and retrieval of a familiar taste. Indeed, mice injected with CNO (n=11) but not saline (n=10) show an impaired neophobic response on day 1 (unfamiliar taste) and an increase in the neophobic response on the next (CNO-free) day. When tested on day 7 (when the taste is now familiar), CNO has no effect indicating that recognition and retrieval of a familiar taste is intact. However, on day 7, the group sizes have been reduced to CNO (n=6) and control (n=5). Why are these group sizes different if the same animals were tested? And if these are different mice, why?

Though we used a concentration of CNO considered to be specific, we thank the reviewers for this constructive comment and preform an additional experiment to validate it. Hence, we added a control group, in which mice were injected with retro-Cre AAV construct at the mPFC, and AAV8_hEF1a-dlox-mCherry (rev)-dlox-WPRE-hGHp (A), an mCherry construct without DREADD receptor, at the aIC. Animals were injected with CNO prior to novel taste procedure (Figure 4—figure supplementary 1a) to control for CNO effects. We observed no difference between the control-virus injected mice that were treated with CNO and the aIC-to-mPFC inhibitory DREADDs-injected mice. This rules out the possible involvement of CNO itself on the measured taste behavior.

Regarding the changes in number of mice in the different procedures- The first experiment was done with an n number of (CNO=5, saline=5), without testing the effect on retrieval of familiar taste. In the second batch, we used another set of animals (CNO=6, saline=5), where we wanted to increase the n number of our experiment, keeping in mind we wanted to control this time for taste recognition. Since, there is no difference between the groups, we did not repeat this test.

4. ACh release in IC is increased following consumption of an unfamiliar taste. The authors showed that novel taste consumption increased excitability in mPFC-projecting neurons in the IC and this excitability was reduced in scopolamine injected mice. However, the authors did not test the mPFC-to-IC pathway. I imagine that the authors would not expect increased excitability in this pathway if its role is limited to expression of neophobia and not learning. But, unfortunately, without this complementary experiment, it remains unclear. Following the same logic, it is unknown if this reduction in excitability is specific to blocking ACh receptors, even if one might expect that it would be ACh-dependent (and NMDA-independent for example) if the increased excitability truly reflects a learning process.

We thank the reviewer for pointing this out. Accordingly, we have removed all the scopolamine data from the manuscript. The manuscript is now better focused on the main hypothesis and take-home messages are clearer.

It would have been interesting to explore the changes occurring in the mPFC following novel taste experience, however, it is not within the scope of our study. The main focus in our paper was to better understand the changes in the aIC and the circuits involving it, in novel taste experience.

Reviewer #2:

While I am in general enthusiastic about this paper, I have several suggestions that I believe might strengthen the manuscript and provide some clarifications regarding the research topic:

1. The key question is related to the overall interpretation that focuses on salience/novelty detection. I found it difficult to delineate whether the effective circuit manipulations affect the detection of the novel taste or anxiety associated with presentation of this taste, or both. I know that these may not be easy to detangle, but I think that consideration of all of these interpretations is needed to fully understand the behavioral effects during expression of neophobia. This can be addressed to some extent by providing additional behavioral data, for example on latency to approach the new taste, number of approaches or total time spent sniffing the bottle, which would indicate novelty detection (or lack thereof) independently of saccharose consumption. Or with a control group receiving a familiar taste (water) in a novel bottle, etc. The authors may already have performed such control experiments, and if so, I would suggest presenting such data to facilitate interpretation. This issue is especially relevant in view of the findings that impaired "expression of neophobia" can nevertheless result in taste memory formation (mPFC to AIC projection results).

We would like to thank the reviewer for their important feedback and for bringing this issue to our attention.

To address the reviewer’s comment, we sought to test the effect of the aIC-mPFC inhibition on anxiety levels. We targeted aIC-mPFC pathway, and tested the open-field behavior when the pathway is manipulated similarly to other experiments in the manuscript. The amount of time spent in the center or corners of the chamber provide us a measurement of stress and anxiety levels during inhibition of aIC-mPFC circuitry (new Figure 6—figure supplementary 2a-c). Furthermore, we tested the latency towards the novel tastant during aIC-mPFC inhibition as suggested by the reviewer. Our results show that inhibiting aIC-mPFC circuit does not affect anxiety levels as measured in the open field test nor in latency towards the novel taste tested (new Figure 6—figure supplementary 2a-e).

2. I would also find it useful to show the baseline levels of pErk (naïve mice) and after water deprivation only in the AIC and mPFC because some areas have elevated baseline activity or could be sensitive to the deprivation protocol. It would also be helpful to see the total numbers of AAV+ cells /layer (in addition to the pErk/AAV %). This will help estimate the variability of labeling between different animals and the overall number of mPFC-projecting neurons.

We thank the reviewers for the suggestions aimed to extend our analysis.

Indeed, drinking following water restriction affects gene expression such as Arc and cFos in the IC (Inberg et al., 2016). However, in order to control the baseline levels of pERK, both novel and familiar taste groups were water restricted equally during the behavioral paradigm. Also, mice were provided with an equal amount (1 ml) of saccharin in order to prevent variability in volumes consumed in the last day. This ensures the equal effect of dehydration/drinking in both groups and allows comparison of the familiarity-novelty axis only.

Following the reviewers' comment and in order to further clarify the aIC-mPFC anatomical connectivity, we performed quantification of rAAV+ neurons in the aIC and the mPFC and provided the data as a supplementary figure (Figure 1—figure supplement1) to better clarify the variability of labeling between the groups. Number of rAAV+ neurons in the aIC and the mPFC was similar both in novel and familiar saccharin groups, indicating no variability in injections/labeling between the groups.

3. The first figures need to have clear indication of the layers and areas used for quantification.

Thanking the reviewers for the comment. We amended the figures to improve clarity as proposed.

4. It would be helpful to demonstrate (and validate) the CNO effects in slices.

The reviewer raises an important point. We agree that electrophysiological demonstration and quantification of the effects of CNO in slices expressing inhibitory DREADDs is fundamental to the credibility of the results. We have previously shown and validated the effect of CNO in slices (Kayyal et al., 2019) in aIC-to-BLA projecting neurons, using the same viral vectors (rAAV-Cre at the BLA and Cre-dependent DREADDs in aIC).

5. I found two aspects of the work underdeveloped: scopolamine and BLA. I would suggest removing the BLA data from this report because all of it is in the supplement and then suddenly shows in the main Figure denoting the circuit. Strengthening the AIC-mPFC circuit model would suffice in my view. The cholinergic aspect would need much more work to integrate because the slice data are very interesting, yet there are no supporting behavioral or circuit data deepening the understanding of the AIC inner layer cholinergic system in the observed effects. If such link is not easy to establish, I would also tend to omit these data and maybe replace them with the retrieval findings. Alternatively, the discussion on cholinergic mechanisms needs to be much stronger and in tighter connection with the demonstrated circuit.

We thank the reviewers for bringing this idea to our notice. To improve the flow and clarity of the paper, BLA data was incorporated to the main text as suggested by the reviewers. Additionally, scopolamine data was removed from the manuscript.

Reviewer #3:

The Rosenblum lab has conducted an important series of experiments to dissect the causal influence of the aIC-to-mPFC connection in distinct aspects of novel taste learning and its adaptive attenuation with subsequent repeated exposure (familiar taste learning). They have used a combination of retrograde labeling to enable visualization of bidirectional monosynaptic connectivity between IC and mPFC, together with pERK labeling to infer in vivo neuronal activation, ex vivo whole cell electrophysiological recordings, and DREADD manipulations to establish the causality of defined connections at distinct cognitive phases during novel taste learning and the subsequent transition to neophobic attenuation with repeated exposure. Overall, the results make an important contribution to our understanding of the cognitive function and plasticity this important neural circuit.

The conclusions of this paper are mostly well supported by data, but a few aspects could be further clarified:

1. AAV8-EF1a and rAAV-hSyn1 promotors for reciprocal aIC-to-mPFC connectivity studies. The EF1a and hSyn1 promoters drive overlapping expression in a broad diversity of neuronal cell types, which leaves open the possibility that distinct classes projection neuron types may be involved. This is particularly relevant for interpretation of the result of the pERK labeling and DREADD inhibition experiments.

We thank the reviewer for pointing this out. We agree with this comment, while hSyn1 promoter is directed only to neuronal population, EF1a directs a broader cell population. However, in the causative experiments in which we inhibited either aIC-to-mPFC or mPFC-to-aIC circuit, we used a retro-Cre AAV construct: rAAV-hSyn1-chI-EGFP_2A_iCre-WPRE-SV40p(A). meaning the targeting eventually was similar (hSyn1 promoter) both in the correlative and the causative studies.

We know from the literature (Baker et al., 2018; Gehrlach et al., 2020; Greig et al., 2013; Molyneaux et al., 2007; Parnavelas, 2000) that cortical and subcortical projecting neurons are mostly excitatory and not inhibitory neurons (>95%). In addition, our electrophysiological studies show that all neurons patched have pyramidal-neuron-like properties. Hence, the probability of us targeting inhibitory neurons is very small.

2. An alternative explanation for the results in Figure 2 of the novel vs. familiar electrophysiological comparison is that familiar saccharin exposure might decrease intrinsic excitability. Similarly, an alternative explanation for the scopolamine results in Figure 3 is that mAchR activation is required at baseline for maintaining intrinsic properties of aIC neurons. This is especially relevant because although the F/I curves for novel vs. familiar compared to saline vs. scopolamine are remarkably similar, the underlying physiological mechanism appears divergent, given the relative discordances in RMP, rheobase, input resistance, and AP half-width (Figures 2/3 and S2/S3).

In response to this important point raised by the reviewer, we have performed an additional experiment to examine the directionality of novel/familiar taste experience upon aIC-to-mPFC projecting neurons by adding a home-caged group of mice (Figure 2; Figure 2—figure supplement 2). Our results show a similar excitability levels between home-caged group and familiar saccharin group that are different from novel saccharin group, meaning the effect is derived from the novelty of the taste (increase excitability in aIC-to-mPFC projections).

In addition, the scopolamine data was removed as suggested by the reviewers.

3. Previous reports have documented the potential confound on behavioral studies with the use of CNO. Therefore, it would be helpful to confirm whether potential actions of CNO on endogenous neurobiology – independent of its function as a ligand for the DREADD receptor – may be influencing results of key experiments in the manuscript. In particular, it is notable that administration of CNO is associated with a consistent decrease in expression of neophobia across multiple experimental conditions (Figures 5, 6 and S5).

We thank the reviewer for raising this important concern.

Doses of CNO used in all experiments were chosen based on recent publications demonstrating that higher chronic doses of the ligand are metabolized into clozapine, which can affect behavior (Gomez et al., 2017).

To directly address whether CNO administration by itself affect classical neophobia or its attenuation, we performed an additional experiment. Control mice (injected with control virus without DREADDs expression at the IC) were prepared, and similar behavioral taste-tests were performed. Our results indicate that the expression of neophobia is unchanged by CNO administration when using control virus (which does not harbor the receptor). This rule out the possibility that the measured behavior was affected by the CNO itself (Figure 4—figure supplement 1a).

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https://doi.org/10.7554/eLife.66686.sa2

Article and author information

Author details

  1. Haneen Kayyal

    Sagol Department of Neuroscience, University of Haifa, Mount Carmel, Israel
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4429-3514
  2. Sailendrakumar Kolatt Chandran

    Sagol Department of Neuroscience, University of Haifa, Mount Carmel, Israel
    Contribution
    Data curation, Formal analysis, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9805-8096
  3. Adonis Yiannakas

    Sagol Department of Neuroscience, University of Haifa, Mount Carmel, Israel
    Contribution
    Data curation, Formal analysis, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Nathaniel Gould

    Sagol Department of Neuroscience, University of Haifa, Mount Carmel, Israel
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Mohammad Khamaisy

    Sagol Department of Neuroscience, University of Haifa, Mount Carmel, Israel
    Contribution
    Data curation, Formal analysis, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  6. Kobi Rosenblum

    1. Sagol Department of Neuroscience, University of Haifa, Mount Carmel, Israel
    2. Center for Gene Manipulation in the Brain, University of Haifa, Mount Carmel, Israel
    Contribution
    Conceptualization, Supervision, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing
    For correspondence
    kobir@psy.haifa.ac.il
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4827-0336

Funding

Israel Science Foundation (isf 946/17)

  • Kobi Rosenblum

Israel Science Foundation (isf 258/20)

  • Kobi Rosenblum

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors would like to thank all current members of the Rosenblum lab for their help and support, to the veterinary team headed by Barak Carmi and Corina Dollingher and technical team headed by Yair Bellehsen. This research was supported by a grant from the Israel Science Foundation (ISF); ISF 946/17 and ISF 258/20 to KR. HK is a recipient of the Edmond de Rothschild’s scholarship. Graphical illustrations were created with BioRender.com.

Ethics

Animal experimentation: All experiments and procedures conducted were approved by the University of Haifa Animal Care and Use committee under Ethical license 554/18 and were in accordance with the National Institutes of Health guidelines for ethical treatment of animals.

Senior Editor

  1. Laura L Colgin, University of Texas at Austin, United States

Reviewing Editor

  1. Mathieu Wolff, CNRS, University of Bordeaux, France

Reviewers

  1. Shauna L Parkes, CNRS, France
  2. Jelena Radulovic, Northwestern University, United States
  3. Steven A Kushner, Erasmus MC University Medical Center, Netherlands

Publication history

  1. Received: January 19, 2021
  2. Accepted: June 29, 2021
  3. Accepted Manuscript published: July 5, 2021 (version 1)
  4. Version of Record published: July 15, 2021 (version 2)

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© 2021, Kayyal et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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