Insula to mPFC Reciprocal Connectivity Differentially Underlies Novel Taste Neophobic Response and Learning

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.

Determining salient stimuli from the continuously perceived sensory input is a key component for 45 effective learning that has been shaped throughout evolution. The term 'salient' is used in psychology 46 and neuroscience to denote a particularly distinct or important stimulus, or an aspect of it (Yantis & 47 Hillstrom, 1994). Attributing salience to a given cue or stimulus, can be affected by genetics, previous 48 experiences, current psychological and motivational state, stress response, goals and motivation 49 relies on a combination of instinctive responses (genetic elements) and brain processes involved in 62 recalling past experiences to help animals cautiously avoid poisonous food, while taking advantage of 63 food that is deemed safe. Therefore, when animals encounter a taste that is completely new to them, 64 they approach it with cautiousness, a phenomenon called taste neophobia (Lin et al., 2015). This 65 neophobic response is the first line of defense, and therefore an important and evolutionarily 66 conserved behavior, manifesting in reluctance to consume the novel taste, for fear of potentially 67 adverse outcomes. Following this tentative consumption of the novel taste, and in the absence of any 68 8 142 Immunohistochemistry and quantification: 143 For perfusion, mice that underwent behavioral experimentation were deeply anesthetized 144 using isoflurane. When the mice were completely anesthetized, they were perfused  Quantification was done using randomly assigned IDs for individual animals, regardless of 176 treatment. Following quantification, we confirmed treatments, and data were analyzed 177 (Graphpad Prism ®).

178
Behavioral Experiments: 179 Mice were randomly allocated to experimental groups. Group size range estimation was 180 based on previously published results using similar methods, as well power calculations 181 (https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html).

182
Immunohistochemical studies: 183 Mice aged 8-12 weeks were injected with rAAV-hSyn1-chI-mCherry-WPRE-SV40p(A), 184 rAAV construct, at the mPFC (to label IC-to-mPFC projecting neurons) or at the IC (to label 185 mPFC-to-IC projecting neurons). One month later, animals were individually separated and 186 given 5 acclimation days. Next, mice were water deprived for 24 hours, and were given tap 187 water (for novel saccharin group) or saccharin (0.5% dissolved in tap water, for familiar 188 saccharin group) pipettes for 20 minutes sessions each day for 6 consecutive days. On the 7 th day, all animals were presented with 1 ml of saccharin, and perfused 20 minutes later, for 190 immunohistochemical analysis. 191 IC-to-mPFC/ mPFC-to-IC circuit inhibition during novel saccharin exposure, or familiar taste 192 retrieval: 193 In order to target the IC-to-mPFC circuit for inhibition, mice aged 8-12 weeks were  To target mPFC-to-IC projecting neurons for inhibition, the same constructs were used to 203 injections in a different set of mice, with the retrograde-Cre AAV construct being injected in 204 the IC, and the Cre-dependent DREADD injected at the mPFC.

299
The osmolarity was 290 mOsm, and pH was 7.3. The recording glass pipettes were patched 300 onto the soma region of rAAV + pyramidal neurons.

301
The recordings were made from the soma of aIC pyramidal cells, particularly from layer 2/3 302 and Layer 5/6. Liquid junction potential (10 mV) was not corrected online. All current clamp 303 recordings were low pass filtered at 10 kHz and sampled at 50 kHz. Series resistance was 304 compensated and only series resistance <20 MΩ was included in the dataset. Pipette

309
Resting membrane potential (RMP) was measured 10 sec immediately after the beginning of

342
We also proved that activity of these projections is necessary for the acquisition and retrieval 343 of an aversive taste memory, but not for attenuating a neophobic response to a novel, non- In many correlative experimental set-ups, it is difficult to dissociate between the cellular 443 activity needed for expressing the behavior from that which is needed to acquire the 444 information for a learning process. Based on the correlation yielded above (Figures 1-3), 445 between novel taste consumption and the reciprocal activation of aIC to/from mPFC, we 446 sought to identify whether activation of the aIC-to-mPFC part of the circuit is necessary for 447 novel taste neophobic responses and/or familiarization learning. We therefore injected mice

466
Our results indicate a necessity for the circuit in denoting the taste as novel, though it does 467 not rule out its effect on other facets such as taste recognition, retrieval or aversion.

468
In order to test recognition and retrieval of a familiar taste, we inhibited the circuit on the 469 seventh day, 1 hour prior to a choice test between saccharin and water, once saccharin 470 neophobia has been attenuated and the taste is no longer novel. We observed no significant 471 difference between the treatment (n=5) and control (n=6) groups (unpaired t-test: t=0.2364, but is vital for both the initial neophobic response, as well as incidental memory formation. 483 We have previously shown that activity within aIC-to-BLA projecting neurons is not This indicates that the mPFC plays a major role in novelty gating, while the detection of novelty and 612 storage of familiar taste memories are both mediated by the aIC. We show that aIC-to-mPFC 613 projections need to be activated during novel taste exposure in order to form a safe taste memory 614 trace, while aIC activation from the mPFC is necessary in order to evoke appropriate behavioral 615 responses to novelty. We thus causally demonstrate that specific connectivity within the aIC and the 616 mPFC allows the identification, the learning and reaction to novel taste stimuli with the memory 617 being formed in the aIC. However, this does not exclude the involvement of other additional stimuli 618 or other circuits that were not the focus of the current study. 619 The entire IC is an integrative hub for saliency processing. We recently demonstrated that the 620 Together, our findings suggest that aIC connectivity to different brain areas differentially conveys the 632 novelty state (aIC-to-mPFC/BLA), while following learning-induced plasticity, a now modified 633 circuit state underlies the encoding of the familiarized taste (aIC-to-mPFC but not to BLA) (Figure 7). Detecting, responding and remembering salient information is a vital property of animal behavior. We 641 show here, for the first time, that the interplay between the aIC and the mPFC in the mammalian brain 642 grasps within it novelty related data, as part of a salience network. Both aIC-mPFC and aIC-to-BLA 643 reciprocal connections are necessary for exhibiting a neophobic response, however, the aIC-mPFC is 644 necessary also to form a memory for a familiar safe taste. In addition, the acquisition and retrieval of 645       to novel saccharin (first exposure) and water choice test, while aversion was assessed.

1114
In the following days, mice were given a choice test without intervention.                       c. d. d. e.