Noradrenaline causes a spread of association in the hippocampal cognitive map

Domain: biology
Published: 2026-03-14
Editorial outcome: major_revision

Abstract
The mammalian brain organises knowledge about entities in the world and relationships between them using cognitive maps. When forming a cognitive map, there is a necessary trade-off between extending the map to make novel inferences, and storing a veridical copy of past experience. However, the neural mechanisms that control this trade-off remain unknown. Using a cross-scale approach that combines a pharmacological intervention in humans with neural network modelling, we show that the neuromodulator noradrenaline elicits a significant ‘spread of association’ across hippocampal cognitive maps. This neural spread of association can be explained by changes in synaptic plasticity that predict overgeneralisation in behaviour. Thus, elevated noradrenaline during learning increases the ‘smoothing kernel’ for plasticity across the cognitive map, allowing disparate memories to become linked and distorted.

Introduction
The mammalian brain stores the relationships between items and events that an organism experiences. This relational information is represented by the hippocampal system, as a cognitive map of the environment1,2. Following its formation, a cognitive map can be used to recall information from the past, while also providing a basis to predict the consequences of upcoming actions, to guide future behaviour3,4. Critically, a cognitive map can extend beyond direct experience, representing more than the sum of its parts by including inferred relationships between stimuli or events in the world5–8. These inferred relationships can be used to draw associations that we have not directly experienced5. For this reason, cognitive maps are credited with enabling adaptive and flexible behaviours, such as inferential choice, generalization and abstraction3.

Yet, there is a fine balance to be struck when building a cognitive map. On the one hand, a cognitive map can support our ability to make highly flexible decisions, by drawing new shortcuts and inferences that extend beyond direct experience5,9,10. For example, Alice may supervise two PhD students, Bob and Charlie. If Bob is going to a conference, I can infer that Charlie may be going too. On the other hand, by drawing relations that deviate from reality, a cognitive map has the propensity to enable overgeneralisation by introducing potentially unwanted distortions in memory11–13. An example of overgeneralisation would be if I see Alice and Bob at a conference, and later ‘recall’ that Charlie was there as well, even though he did not in fact attend. When forming a cognitive map, the relative ‘spread of association’ across nodes in the cognitive map may critically determine whether the map facilitates adaptive or maladaptive behaviour.

Here we ask: what is the neural mechanism that titrates the spread of association within the cognitive map? One candidate mechanism for titrating the ‘spread of association’ within the cognitive map involves the neuromodulator noradrenaline14–16. Noradrenaline profoundly alters plasticity in the brain by reducing the induction threshold for synaptic plasticity and reducing activity in inhibitory interneurons17–23. Noradrenaline may therefore titrate the spread of association within the cognitive map by regulating the extent to which synaptic plasticity links discrete memories together.

To test this hypothesis, here we use a synergistic cross-scale approach where we apply the same behavioural paradigm in both a computational model and in human participants who undergo an fMRI scan with a double-blind pharmacological intervention. We predicted that learning during elevated noradrenaline should increase the spread of association in the underlying cognitive map.

Results
Experimental design
To test this prediction, in humans we manipulated noradrenaline during a learning task using a between-subject, double-blind, randomised placebo-controlled design. At the start of the experiment, participants were given either a single dose of the noradrenergic reuptake inhibitor, atomoxetine (‘ATX’), or a placebo (‘PLC’). Previous studies demonstrate that atomoxetine can be used to increase levels of noradrenaline throughout the brain24,25. Our blinding procedure was effective and participants in the drug group (ATX) were unable to correctly report whether they received atomoxetine or placebo (Supplementary Fig. 1, Supplementary Table 1).

After receiving atomoxetine or placebo, participants waited 90 minutes to allow noradrenaline levels to reach peak plasma concentration26. Participants then completed a learning task. We designed the task such that the underlying task structure was susceptible to overgeneralisation and memory distortion. The task required participants to learn a set of pairwise associations between bird stimuli that could be arranged in a ring structure (Fig. 1a). Each bird stimulus was paired with two other bird stimuli, with each pair of birds appearing in a unique context (Fig. 1b-c, Supplementary Fig. 2). For example, bird 2 appeared both with bird 1 (in room A) and with bird 3 (in room B). Participants were not made explicitly aware of the underlying task structure, including the ring topology. Memory errors between bird stimuli and contextual cues provided a means to later test participants’ susceptibility to overgeneralise and distort the relational task structure.

Participants learned to associate pairs of birds using a three-alternative forced choice task (‘Learning task’, Fig. 1d). During the learning task participants were not tested on the relationships between the bird stimuli and the contextual cues, leaving these relationships implicit. All participants learned the pairwise associations with high accuracy and there was no significant difference in learning between the ATX and PLC groups (Fig. 1e-f, Supplementary Fig. 3b).

Importantly, this task design allowed us to test whether elevated noradrenaline introduces excess inference or a ‘spread of association’ across the underlying cognitive map. Specifically, we predicted that if noradrenaline increases excitatory plasticity between nodes in the underlying cognitive map (Fig. 1a), stimuli and contextual cues situated in proximal locations on the ring may become erroneously associated due to a ‘spread of association’. By contrast, those situated at distal locations on the ring should remain distinct, thus providing the necessary control for those in proximal locations. Our experimental design thus provided a precise and controlled means to test evidence for the effect of noradrenaline on forming a cognitive map, at both a behavioural and neural level.

Behavioural evidence for overgeneralisation with noradrenaline
Four days after learning (Day 5, Fig. 1g), when the effects of atomoxetine had washed out26, participants returned to perform memory tests designed to provide behavioural tests for overgeneralisation across memories. We included memory tests for both the pairwise associations between birds learned on day 1 (‘Explicit memory test’, Fig. 2a; ‘Ring Topology test’, Supplementary Fig. 4a) and the incidental pairings between the bird stimuli and contextual cues (‘Implicit memory test’, Fig. 2b; ‘Ring Topology test’, Supplementary Fig. 4a).

For behavioural tests on day 5 where overall accuracy in both groups was relatively high, we observed no significant group difference in explicit or implicit memory accuracy (ATX vs. PLC: Explicit memory: Fig. 2c, Supplementary Fig. 4b; Implicit memory: Fig. 2d, Supplementary Fig. 4c). Therefore, we observed no evidence to suggest that learning under elevated noradrenaline quantitatively strengthens memory encoding or consolidation of component associations.

However, for the ‘Implicit memory test’, where overall accuracy in both groups was relatively poor, the error trials (74% of trials, on average) afforded an opportunity to test evidence for overgeneralisation and distortions in memory.

Specifically, we reasoned that if the underlying memory map is distorted to include a ‘spread of association’ around the ring, participants should be more likely to overgeneralise by associating bird stimuli with incorrect contextual cues that are proximal rather than distal on the ring. For example, for bird 1, rather than selecting room A where bird 1 and bird 2 were located, participants made an overgeneralisation error if they selected room B, where bird 2 and bird 3 were located. We hypothesised that learning under noradrenaline should increase these overgeneralisation errors, due to the spread of association in the underlying cognitive map. Consistent with this prediction and regardless of the precise mathematical definition of overgeneralisation (see Methods), the ATX group compared to PLC was significantly more likely to erroneously report associations between proximal rather than distal stimuli on the ring (Fig. 2f-g; Supplementary Fig. 4d). These results suggest that learning under elevated noradrenaline increases the spread of associations between stimuli that were never presented together but are located nearby in a cognitive map.

Physiological markers of elevated noradrenaline
In clinical trials substantial interindividual variability is observed in response to neuropsychiatric medication, such as atomoxetine. Here, we measured this variability to quantify interindividual differences in the response to drug in the ATX group. We used two physiological markers of noradrenergic arousal, namely the pupil dilation response, and cortical measures of GABAergic tone.

Pupil response to oddball stimuli is elevated under atomoxetine
We collected pupillometry data after the learning task during an “oddball” detection task performed inside the MRI scanner. Work in animals and humans shows that increases in pupil diameter reflect increases in the availability of noradrenaline28–32. In line with the suggestion that noradrenaline signals ‘unexpected uncertainty’ 33, both a phasic and tonic component of the pupil dilation response are typically observed in response to an unexpected “oddball” stimulus34–36. While the phasic component involves a transient deflection from baseline, the tonic component is superimposed on the phasic component and involves a more gradual shift in baseline response that can last for several seconds to minutes36. The phasic component has been related to phasic bursts of locus coeruleus (LC) activity, while the tonic component has been related to baseline levels of noradrenaline29,37 (and is reported to increase with ATX38–40.

Here, we used the pupil response to “oddball” stimuli as a physiological marker for noradrenaline. On each trial in the oddball detected task, participants were presented with either a familiar stimulus (91% of trials) previously observed during the learning task, or an oddball stimulus (9% of trials, Fig. 3a). Participants were required to make a button press response when they detected an oddball stimulus. We analysed pupil dilation following stimulus presentation. Consistent with previous studies34–36,41,42 in both the ATX and PLC groups there was an enlarged phasic pupil response following oddball stimuli. The tonic component of this oddball response was greater in the ATX group compared to the PLC group, with a significant group difference from ~6-10 s after stimulus onset (Fig. 3b). Thus, the ATX group showed an increase in their sustained tonic pupil dilation response to unexpected stimuli, providing a physiological maker for the drug response of individuals within this group.

Elevated noradrenaline decreases inhibitory tone in neocortex
Having established that a single dose of atomoxetine increases pupil response to surprising stimuli, we tested evidence for a second physiological marker of elevated noradrenaline, namely cortical inhibition. The majority of noradrenergic projections to cortex originate in the LC and stimulation of LC in rodents induces a decrease in cortical inhibitory tone20. In humans, a single dose of atomoxetine reduces paired-pulse suppression using visual evoked potentials in visual cortex, indicating an increase in cortical excitability43. Taken together, we predicted that elevating noradrenaline using a single dose of atomoxetine should induce a reduction in cortical inhibition, to shift cortical activity in the direction of increased excitability.

To measure cortical inhibition in our participants, we used Magnetic Resonance Spectroscopy (MRS) (Fig. 1g) to quantify the local concentration of different neural metabolites in the brain, including the concentration of cortical gamma-aminobutyric acid (GABA+, GABA and macromolecules) together with Glx (glutamate and glutamine). We measured GABA+ from a voxel positioned in the Lateral Occipital complex (LOC) (Fig. 3c), a brain region implicated in visual object processing, including basic object recognition, scene perception and processing of object shape44,45. Indeed, during the scan task, the BOLD response in LOC was significantly modulated by task stimuli (Supplementary Fig. 5b). In response to ATX, any change in Glx/GABA+ in LOC should therefore result in task-relevant neural changes.

In line with previous evidence from rodents20, GABA+ in LOC was significantly reduced in the ATX group compared to the PLC group (Fig. 3e). Moreover, this reduction in GABA+ in the ATX group accounted for a significant increase in the ratio between Glx and GABA+ in the ATX compared to the PLC group (Glx:GABA+, Fig. 3f)46. Critically, there was no significant difference in MRS quality metrics between the ATX and PLC groups (Supplementary Fig. 5c-e). When measuring GABA+ and Glx:GABA+ ratio from V1, no significant difference between ATX and PLC was observed (Supplementary Fig. 6c-d). The lack of effect in V1 may be explained by lower Signal to Noise Ratio (SNR) in V1 compared to LOC, as indicated by significantly lower temporal SNR (tSNR) in this region (Supplementary Fig. 6g). In LOC, significant evidence for reduced GABA+ in ATX compared to PLC suggests that a single dose of atomoxetine reduces cortical inhibition in brain regions sensitive to task stimuli, namely the visual object processing region LOC. Moreover, taking Glx:GABA+ as a proxy measure of the balance between cortical excitation and inhibition (E/I), these results suggest that noradrenaline shifts participants’ E/I ratio along a continuum in the direction of increased cortical excitability. Taken with the pupil dilation response, this MRS measure of reduced cortical inhibition or E/I ratio in ATX provides a second physiological marker of the response to drug.

Reduced inhibitory plasticity during learning leads to spread of association in neural network model
Having confirmed that a single dose of atomoxetine induces expected changes in physiological markers of noradrenergic arousal, we next investigated the effect of elevated noradrenaline on the formation of a cognitive map of the task. At a behavioural level, an increase in overgeneralisation was observed in ATX but not PLC (Fig. 2f,g). Notably, no direct relationships were observed between the physiological measures of noradrenergic arousal and these behavioural measures of overgeneralisation (Supplementary Fig. 7). We therefore asked whether physiological markers of noradrenergic arousal provide a precise predictor for neural changes in the underlying cognitive map, which in turn predict behavioural measures of overgeneralisation.

To address this, we first asked whether this behavioural evidence for overgeneralisation in the ATX group can be accounted for by distortions in the underlying cognitive map, i.e. the neural representation of the task. Specifically, we predicted that learning under elevated noradrenaline should introduce ‘leaky’ excitation around the ring structure leading to more pronounced plasticity between proximal compared to distal nodes and establishing a spread of association in the underlying memory map. To test this hypothesis, we used a combination of computational and experimental approaches.

We built a proof-of-concept computational model to probe whether learning during a period of reduced inhibition alters synaptic modification to allow for a spread of association in the underlying cognitive map. To test this hypothesis, we refined a set of previously published spiking neural network models47–49 and utilised a co-dependent plasticity rule formalised using multiple empirical findings47. The co-dependent plasticity rule was applied to all excitatory synapses projecting onto excitatory cells (E-E), and all inhibitory synapses projecting onto excitatory cells (I-E). For simplicity, all remaining synapses were held static after their values were initialised, and manual changes in synaptic weight were applied to form well-defined cell-assemblies and embed the underlying ring structure.

Using our model, we replicated our experiment using a reduced ring of associations in silico. To represent distinct stimuli, we first embedded six cell assemblies, or ‘nodes’ 1–6, that were balanced by local intra-nodal inhibitory plasticity. To simulate the memory map in this pre-learning state (prior to learning the ring of associations), we externally activated one independent cell assembly ‘1’ (to simulate sensory input) and found no change in the activity of other nonoverlapping assemblies (Fig. 4a).

Next, we simulated the consequences of learning the ring of associations, by manually embedding the underlying ring structure into the network. Excitatory (E-E) connections between neighbouring cell assemblies were strengthened, and resultant surplus excitation was balanced by strengthening relevant inhibitory (I-E) connections between neighbouring nodes, enabling clear identification of distinct excitatory and inhibitory connections associated with each assembly. Together, this resulted in the formation of a ring structure, with each node being connected to two other nodes via both excitatory and inhibitory connections (PLC network: Fig. 4b, left). After embedding the underlying ring structure, the co-dependent plasticity rules ensured overall network stability despite external perturbations in the excitatory-inhibitory balance. Importantly, despite strong excitatory connections between neighbouring assemblies, no significant co-activation was observed along the ring structure when externally activating one cell assembly, due to the proportionally strengthened inhibitory connections (PLC network: Fig. 4b, middle-right).

In the computational model, we then simulated the effect of learning under elevated noradrenaline (‘atomoxetine’). Unlike in the placebo model, in the atomoxetine model the associations were learned under a regime of reduced global inhibitory firing across the network. Consequently, in the atomoxetine model, local inhibitory (I-E) connections between nodes were only minimally strengthened in response to learning (ATX network: Fig. 4c, left). This resulted in an excitatory surplus which was not balanced by synaptic inhibition, leading to elevated excitatory plasticity. These changes manifested as a gradient of co-activation along the ring structure in response to activation of a single cell assembly ‘1’ (ATX network: Fig. 4c, middle-right). The graded co-activation occurred relative to their respective distances from assembly ‘1’, with greater co-activation of nearby assemblies (‘2’ and ‘6’) in contrast to more distal assemblies (‘3’ and ‘5’, and ‘4’). This graded co-activation further led to graded plasticity across cell assemblies, indicated by the graded changes in synaptic weights between pairs of cell assemblies (Fig. 4c, middle-right). Thus, in our proof-of-principle model, learning under reduced inhibitory firing was sufficient to embed a spread of association in the underlying memory map.

Using our neural network model, we further explored alternative mechanisms that can account for a spread of association in the underlying memory map. Consistent with previous modelling of associative memory maps50, in the post-learning PLC network we show that a reduction in local (weakening only inter-nodal I-E connections) but not global (weakening all network E-I connections) inhibition is sufficient to reveal a spread of association (Supplementary Fig. 9). This reduction in local inhibition may plausibly occur via transient disinhibition during memory recall51,52. However, we note that when memory recall performance was tested on day 5 (Fig. 2; Supplementary Fig. 4), the effect of the drug had washed out, giving no reason to expect a group difference in local inhibition at this time point.

Another possibility is that the spread of association in the ATX group can be attributed to an increase in assembly overlap. Indeed, some degree of both functional and structural assembly overlap is expected across neighbouring assemblies due to associative learning of bird-bird pairs, in line with empirical evidence53,54 and computational models50,55,56. In the context of our model, we explored how assembly overlap influences overgeneralisation. In the PLC network, we found that overgeneralisation is observed when neighbouring assemblies overlap by ≥40%, a value in part determined by network size (Supplementary Fig. 10). However, given that no significant evidence for overgeneralisation was observed in the PLC group (Fig. 2f-g; Supplementary Fig. 4d), we conclude that assembly overlap due to mere associative learning is not sufficient to drive overgeneralisation behaviour. Rather, given that we observe significant overgeneralisation in the ATX group, and a significant group difference (ATX-PLC) (Fig. 2f-g; Supplementary Fig. 4d), this suggests that elevated noradrenaline during learning increases cell assembly overlap. Indeed, this is precisely what we observe (Fig. 4c) where graded co-activity across the network results in graded synaptic plasticity, increasing functional overlap between cell assemblies in a graded manner. Overall, our computational model suggests that when learning occurs during a period of reduced inhibition, local inhibitory rebalancing is compromised, leading to synaptic modifications that embed a spread of association across memories in the underlying cognitive map.

Elevated noradrenaline increases spread of association in the underlying cognitive map
To empirically test the predictions of our model in our participants (Fig. 1-3), we used functional Magnetic Resonance Imaging (fMRI) to measure neural representations of task stimuli and representations of the underlying memory map (Fig. 1g). Previous studies in both animals and humans demonstrate that the hippocampus represents both spatial and abstract cognitive maps of the environment1,3. Thus, in the ATX group, we predicted hippocampal representations that reflect distortions in the overall task structure. In addition, based on our use of an object-in-scene (bird-in-room) paradigm, we predicted neural representation of task cues in visual object and scene processing regions, namely LOC and parahippocampal cortex. Moreover, the parahippocampal cortex provides a major input-output route for information processing between sensory cortex (e.g. LOC) and the hippocampal formation57,58 (Fig. 5a). Analyses were therefore performed across these three regions of interest: LOC, parahippocampus and hippocampus, together with the whole imaged brain volume.

To measure neural representations using fMRI we took advantage of repetition suppression, which relies on the fact that neurons show a relative suppression in their activity when presented with repeated stimuli to which they are responsive59,60. While typically used with fMRI to access sub-voxel representations for single stimuli61, “cross-stimulus” suppression can be used to index the relative co-activation or overlap between neural representations coding for two different stimuli48. To measure cross-stimulus suppression, stimuli were presented in a pseudo-randomized order during the fMRI scan task (Fig. 3a). Each stimulus was categorised post hoc according to the node distance of the stimulus that came immediately before, as defined by the underlying ring structure (Fig. 5b). For example, bird stimuli preceded by an associated bird were categorised as proximal ‘1 node’ trials (e.g. bird ‘1’ preceded by bird ‘2’), while bird stimuli preceded by birds further away on the ring structure were categorised accordingly (e.g. ‘4 nodes’: bird ‘1’ preceded by bird ‘5’) (Fig. 5b). Notably, the symmetrical ring topology of the underlying memory map provided an efficient way to measure node distance from each node.

We reasoned that if noradrenaline leads to a spread of association in the underlying memory map, we should observe greater co-activity, and therefore greater cross-stimulus suppression, for proximal compared to distal nodes. To test this prediction, we first identified brain regions where cross-stimulus suppression scales across all possible node distances. Consistent with our prediction, in the ATX group we observed significant evidence for a spread of association across the underlying memory map in right hippocampus and parahippocampus (Fig. 5c, Supplementary Table 3). No significant effect was observed in the PLC group (Supplementary Fig. 12b). While no significant group differences between ATX and PLC was observed across the hippocampus-parahippocampus region of interest (Fig. 5e), in parahippocampus there was a trend towards a significant group difference (Fig. 5f). These results show qualitative agreement between our data and neural network model.

We next sought to quantitatively compare our data to the output of the neural network model. Notably, as our neural network model was simplified to include only 6 cell assemblies (Fig. 4), we sought to identify brain regions where cross-stimulus suppression scales with node distance as quantified using the simulated firing rates generated by our neural network model in response to stimulating assembly ‘1’ (ATX model: Fig. 4c). Thus, for each node (Fig. 1a) we assessed evidence for a spread of association up to a maximum distance of 3 nodes apart. In the ATX group we again observed significant evidence for a spread of association across the underlying memory map in the right hippocampus (Fig. 5g, Supplementary Fig. 12c, Supplementary Table 4). No significant effect was observed in the PLC group (Supplementary Fig. 12d). While no significant group differences between ATX and PLC were observed across the hippocampus-parahippocampus region of interest (Fig. 5h, Supplementary Table 5), a post hoc test revealed a significant group difference in right hippocampus (Fig. 5h-i). Taken together, these results support the predictions from the model by suggesting that learning under elevated noradrenaline leads to a spread of association in the underlying cognitive map.

As the response to the drug was variable in the ATX group, in subsequent analyses of the underlying neural representations we asked whether this variance could be explained by behavioural measures and our two physiological markers indicating individual differences in the response to the drug.

First, we asked whether the neural spread of association in the underlying memory map observed immediately after learning (Fig. 5d,g) can predict overgeneralisation in behaviour on day 5 (Fig. 2f-g). We reasoned that participants who show stronger neural spread of association in their underlying memory map immediately after learning should retain this knowledge and show stronger evidence for spread of association in subsequent behaviour. In the ATX group, we observed a significant positive relationship between the neural measures of spread of association in hippocampus and parahippocampal cortex and overgeneralisation errors reported in behaviour using two different behavioural measures (Fig. 6a-b). The significant peak of the effect was observed in hippocampus, verified using post hoc tests (Supplementary Table 6, Supplementary Table 7). Therefore, participants who showed a greater spread of association in the underlying memory map on day 1 went on to show a greater spread of association in their behaviour on day 5. Notably, no significant effects were observed for the PLC group (Supplementary Fig. 13a,b) and a significant difference was observed between the ATX and PLC groups, again using two different behavioural measures (Fig. 6c-d, Supplementary Table 8, Supplementary Table 9). These results suggest that learning under elevated noradrenaline leads to a spread of association in the underlying cognitive map that manifests as a systematic and sustained overgeneralisation in the behavioural readout of memory.

Physiological markers of noradrenaline predict neural spread of association
These findings are observed after only a single dose of atomoxetine with variable effect across individuals. Importantly, here we measured the individual response to drug using two physiological markers of noradrenergic arousal, namely the significant increase in pupil dilation response to surprising stimuli (Fig. 3b) and the significant reduction in GABAergic tone in LOC (Fig. 3e). Using these two physiological markers as a measure for the response to drug, in the ATX group we asked whether individual differences in the neural spread of association in parahippocampus-hippocampus measured using fMRI can be predicted by the physiological response to drug.

Within the ATX group, we observed a significant negative relationship between GABA+ in LOC and the neural spread of association in parahippocampus-hippocampus (Fig. 6e). The peak of the effect was observed in parahippocampus, verified using post hoc tests (Supplementary Table 10). Notably, there was no significant evidence for this relationship in the PLC group (Supplementary Fig. 13c), though no significant group difference was observed (Supplementary Fig. 13d). Therefore, participants in the ATX group who learned the memory map with lower GABA+ levels in LOC showed stronger neural spread of associations in their underlying memory map. This suggests that the spread of association in the underlying memory map can be attributed to learning in a state of reduced inhibitory tone, which differentially affects plasticity between proximal compared to distal nodes on the ring.

We next asked whether our second physiological measure of response to drug, namely the pupil response to surprising stimuli, could also predict the neural spread of association in parahippocampus-hippocampus. Within the ATX group, we observed a positive trend between the pupil dilation response and the neural spread of association in parahippocampus-hippocampus (Fig. 6f). The peak of the effect was significant in hippocampus, verified using post hoc tests (Supplementary Table 11). Therefore, participants in the ATX group who showed a stronger pupil response also showed a stronger neural spread of association in their representation of the underlying memory map in hippocampus. No significant relationship was observed in the PLC group (Supplementary Fig. 13e) and significant group differences were observed in both hippocampus and parahippocampus, verified using post hoc tests (Supplementary Fig. 13f, Supplementary Table 12). Taken together, these results suggest that the significant neural spread of association in the underlying memory map of the ATX group can be predicted by variance in the response to drug. Moreover, a significant between group difference is observed for this relationship when assessing variance in the response to drug captured by the pupil dilation response.

Methods
Participants and double blinding
44 healthy volunteers participated in the study. We implemented a double-blind, randomized, placebo-controlled study design. Participants were randomly assigned to one of two groups which were stratified by sex: one group who received a single dose of 10mg atomoxetine (ATX group, n=22, mean age: 23.9 +/- 5.23 yrs, 11 women) and one group who received placebo (PLC group, n=22, mean age: 25.2 +/- 4.80 yrs, 11 women). Participants reported no history of psychiatric disease, and were not taking any prescription medicines within the 2 weeks prior to the time of testing, with the exception of the oral contraceptive pill. All experiments were approved by the University of Oxford ethics committee (reference number R60579/RE004). All participants gave informed written consent.

Randomisation was performed by a researcher not involved in recruitment and data collection (LC). The drugs were administered by an experimenter (RK), who was blind to the drug condition. At the end of the final testing day, participants and experimenter indicated whether they thought the participant had received atomoxetine or placebo.

The effectiveness of the double blinding procedure was assessed using the Bang’s Blinding Index (BI) 75. A score of 1 indicates that all responses were correct and complete unblinding is inferred; a score of -1 indicates that all responses were incorrect and complete blinding is inferred or unblinding in the opposite direction (e.g. opposite guessing); a score of 0 indicates that half the guesses were correct and half of the guesses were incorrect, inferring successful blinding with random guessing. Scores between -0.2 and 0.2 indicate that blinding was successful.

Behavioural training
On the first day of the experiment, all participants (n=44) were given an over-encapsulated capsule, which contained either a single dose of 10mg atomoxetine (ATX group) or an empty capsule (PLC group). Participants were then required to wait for 90 minutes (Fig. 1g) to allow time for the drug to take effect 26.

Following the waiting period, participants learned 11 pairwise associations between bird stimuli. Each pair of birds was presented in a particular context, where the context constituted a living room interior (Fig. 1b,c). The bird stimuli were always presented in the same location within each particular context (Supplementary Fig. 2). Together, the context dependent pairwise associations formed a symmetrical ring structure (Fig. 1a). This ring structure provided an efficient way to later measure representational overlap between proximal and distal associative links as each bird could be used as a control for an association on the other side of the ring (see fMRI scan task). Participants were not made aware of the underlying ring structure.

To learn the bird-bird associations, participants performed a 3-alternative forced choice task with feedback (‘learning task’, Fig. 1d). Every trial, participants were shown a background stimulus with one of the bird stimuli in place. Choice stimuli could be displayed upon pressing the buttons ‘b’,’n’, or ‘m’ on the keyboard and participants confirmed their choice by pressing one of these three buttons together with the spacebar. There was no response deadline. Feedback was subsequently presented for 1.5 s, after which the background with the correct stimulus pairing was shown until the participant again pressed spacebar to proceed to the next trial. Participants were required to score >90% correct on the 3-alternative forced choice learning task for two consecutive blocks. Upon reaching this criterion, participants completed the ‘Inference task’ (Supplementary Fig. 3a) which involved participants indicating which bird stimulus shared an association with the probe bird (e.g. probe bird: bird ‘1’; inferred choice: bird ‘3’) thus making trajectories along the ring structure. The inference test was performed in the absence of contextual cues or feedback, again using a 3-alternative forced choice test. Importantly, throughout these training tasks the bird-bird associations were made explicit while the bird-sofa relationships remained implicit.

Four days later, on Day 5, participants completed a battery of tests to assess memory for explicitly and implicitly learned associations. The battery of tests included the following: (1) ‘Explicit Memory Test’, where a 3-alternative forced choice task was used to test memory for bird-bird associations in the absence of contextual cues or feedback (Fig. 2a); (2) a repeat of the ‘Inference Test’, as encountered on day 1 and described above (Supplementary Fig. 3a); (3) ‘Implicit Memory Test’, where a 6-alternative forced choice task was used to test memory for bird-context associations in the absence of feedback (Fig. 2b); (4) ‘Ring Topology Test’, where participants were given the opportunity to construct the ring topology by placing each bird pairing in the correct scene using drag-and-drop (Supplementary Fig. 4a), completed after all other tests. The ‘Ring topology test’ was collected in all participants apart from the first, resulting in n=43 for this task.

In the Implicit Memory Test images of sofas taken from the background interiors were used as contextual cues, rather than the full image of the living room interior. Importantly, throughout the task the bird stimuli were always presented in the same location within their contextual interior but never on or proximal to the sofas.

For each test, average accuracy was estimated for each participant. On the ‘Implicit Memory Test’ (Fig. 2b), as overall memory accuracy was close to chance (Fig. 2d), we analysed the error trials to estimate two behavioural measures for the relative spread of association between stimuli. The first measure sought to quantify ‘Overgeneralisation errors’, defined as: 

𝑂𝑣𝑒𝑟𝑔𝑒𝑛𝑒𝑟𝑎𝑙𝑖𝑠𝑎𝑡𝑖𝑜𝑛 𝑒𝑟𝑟𝑜𝑟𝑠= ( (∑𝑛 𝑡𝑟𝑖𝑎𝑙𝑠( 𝑝𝑟𝑜𝑥𝑖𝑚𝑎𝑙 𝑐ℎ𝑜𝑖𝑐𝑒 𝑡𝑟𝑖𝑎𝑙 ) / 𝑁 𝑝𝑟𝑜𝑥𝑖𝑚𝑎𝑙 𝑐𝑢𝑒𝑠) - (∑𝑛 𝑡𝑟𝑖𝑎𝑙𝑠( 𝑑𝑖𝑠𝑡𝑎𝑙 𝑐ℎ𝑜𝑖𝑐𝑒 𝑡𝑟𝑖𝑎𝑙 ) / 𝑁 𝑑𝑖𝑠𝑡𝑎𝑙 𝑐𝑢𝑒𝑠) ) / (𝑁 𝑡𝑟𝑖𝑎𝑙𝑠 𝑤𝑖𝑡ℎ 𝑜𝑝𝑝𝑜𝑟𝑡𝑢𝑛𝑖𝑡𝑦 𝑓𝑜𝑟 𝑜𝑣𝑒𝑟𝑔𝑒𝑛𝑒𝑟𝑎𝑙𝑖𝑠𝑎𝑡𝑖𝑜𝑛 𝑒𝑟𝑟𝑜𝑟) * 100% ( 1 )

i.e., the percentage of trials where participants erroneously linked a bird stimulus to a neighbouring (‘proximal’) contextual cue in the underlying ring structure (Fig. 2e, e.g. selecting sofa B when prompted with bird 1), minus the percentage of trials where any other ‘distal’ error was made (e.g. selecting sofa E when prompted with bird 1), divided by the total number of trials where there was the opportunity for overgeneralisation errors. The second measure sought to measure ‘Mean rank proximity’, defined as: the average distance in links between the probe and the erroneously chosen contextual cue. For instance, given probe ‘1’, a choice for sofa ‘B’ would be ranked as ‘2’ whilst a choice for sofa ‘D’ would be ranked as ‘4’. Participants with a high ‘Overgeneralisation error’ score and a low ‘Mean rank proximity’ score therefore showed a tendency to select incorrect contextual cues that were proximal rather than distal relative to the probed bird stimulus.

In the ‘Ring Topology Test’, we further test behavioural evidence for spread of association between stimuli using a metric of ‘Topological Distance’ which we define as: the average link distance between the bird-bird and bird-scene choices made by each participant. For example, a correct pairing between bird ‘1’ and ‘2’, or between bird ‘1’ and sofa ‘A’ would be given a rank score of 1, while an incorrect pairing between bird ‘1’ and bird ‘3’, or between bird ‘1’ and sofa ‘B’ would be given a rank score of 2, and so on. Thus, if the mean ‘Topological distance’ score is low, participants are more likely to erroneously position proximal rather than distal stimuli as neighbours on the rink.

MRS data collection and analysis
MRS spectra were acquired using a locally developed version of the CMRR Spectroscopy Package MEscher-GArwood Point RESolved Spectroscopy (MEGA-PRESS) sequence in two 2 x 2 x 2 cm3 VOIs, with TE = 68 ms, TR = 1.5 s, flip angle 90 deg, ON editing pulse 1.9 ppm, OFF editing pulse 7.5 ppm. Water suppression was achieved using per-subject calibrated VAPOR and dual-band MEGA editing. Outer volume suppression around each axis was interleaved with the water suppression module. Unsuppressed water signal was collected for internal water referencing. The first volume of interest (VOI) was centered bilaterally on the calcarine sulcus in visual cortex (V1). The second VOI was positioned in right LOC. For each measurement 320 spectra were acquired (160 edit-ON and 160 edit-OFF), resulting in an acquisition time of about 8 min for each VOI. Shimming was performed using the vendor’s own algorithm, based on GRE B0 maps (GRE Brain)76.

Glx and GABA+ concentrations were quantified using Gannet version 3.1.4 77, with water used as a reference. Gannet’s standard preprocessing pipeline was used, which includes frequency and phase correction by spectral registration and line broadening. Individual ON and OFF subspectra were then averaged and edited spectra were generated by subtracting the averaged edit-OFF spectra from edit-ON spectra. Notably, the editing approach not only targets GABA but also other macromolecules at 3ppm, therefore the concentrations of GABA+ (GABA and macromolecules) are reported. Furthermore, the editing approach also targets nearby glutamate/glutamine resonances, providing an estimate of Glx concentration. However, it should be noted that the MEGA-PRESS uses parameters optimised for editing GABA and not glutamate. Therefore, compared to quantifying Glx using a PRESS sequence, test-retest reliability of the Glx is likely compromised when estimated with MEGA-PRESS 78,79. Metabolite concentrations were quantified in pseudo-absolute molality units (approximating moles of GABA per kg of solute water) and relaxation and tissue-corrected 80. Grey matter, white matter and CSF tissue fractions for determining tissue-corrected concentrations were obtained for both using SPM12.

Reliable model fits were achieved for 38 out of 41 V1 acquisitions (ATX: 20, PLC: 18) and for 37 out of 43 LOC acquisitions (ATX: 19, PLC: 18). In data where no reliable model fit was achieved, GABA+ was inestimable due to lipid contamination or low SNR. Data quality was quantified using the signal-to-noise ratio (SNR) and full-width-at-half-max (FWHM) of N-acetylaspartate (NAA) and fit error of the GABA+ peak provided by Gannet (Supplementary Fig. 5). The average GABA+ and Glx concentrations for the ATX and PLC groups were compared using two-tailed permutation tests. A null distribution of the expected difference was generated by permuting group labels (ATX, PLC) 10,000 times using MATLAB’s random number generator.

fMRI scan task
During the fMRI scan, participant viewed the 11 bird stimuli encountered during learning, across 4 scan blocks. During 2 of these scan blocks, bird stimuli were interleaved with the 11 sofa contextual stimuli. Stimuli were presented via a computer monitor inside the scanner bore. On each trial, stimuli were presented for 1000 ms (Fig. 3a). The inter-trial interval was selected from a truncated gamma distribution with a mean of 2.3 s, minimum of 1.3 s and maximum of 14 s. To control for potential confounding effects of expectation suppression 81, all possible transitions between non-repeating stimuli were presented once in each block, in a fully randomized order. Participants performed a task incidental to the contrast of interest which involved identifying whether the presented stimuli were familiar or ‘‘oddball”. Oddball stimuli were distorted or differently coloured versions of the stimuli encountered during training, and were randomly inserted into 9% of trials. Participants were instructed to press a button on an MR compatible button box using their right index finger when they identified “oddball” stimuli, but not if the stimulus was familiar. Participants received a monetary reward for each correct oddball identification. No feedback was given other than the total monetary reward after each block.

Pupillometry acquisition, preprocessing, and analysis
During the fMRI scan task (see fMRI scan task) pupillometry data was collected. We measured pupil size using an EyeLink eyetracker (SR Research) sampled at 500 Hz. Pupillometry data was preprocessed using a standardised processing pipeline. Briefly, data was smoothed using a 120ms Gaussian kernel, blinks were identified and removed by interpolating the pupillometry data across the blink period. Data were epoched around presentation of the visual stimuli. Trials on which the pupil measurement was lost for a period of >1000ms were removed from the analysis. Participants were excluded from subsequent analysis if more than 50% of trials had been removed (n=10 participants). Overall, pupillometry data from 34 out of 44 participants was included (ATX: 15, PLC: 19), with an average of 672.50 ± 17.69 ‘regular’ trials and 58.82 ± 1.36 ‘oddball’ trials (mean ± SEM).

To normalise pupil size for each scan block we expressed pupil size in terms of the percentage difference from the average pupil size across the block. To account for changes in pupil size over the course of the fMRI scan task, each epoched pupil response was baseline corrected. The baseline was defined as the mean of the normalised pupil size in the 250 ms window prior to stimulus presentation. To assess the effect of surprise on the pupil response, for each participant we computed the difference between the epoched pupil response for ‘oddball’ vs ‘regular’ stimuli. To assess the difference in pupil response between the ATX and PLC groups, we used a cluster-based significance approach where we permuted subject labels x10,000 for each timepoint, to define the size threshold for a statistically significant cluster (p<0.05)82,83.

fMRI imaging protocol
The fMRI scan task was performed inside a 3 T Magnetom Prisma (Siemens) using a 32-channel head and neck coil (Nova Medical Inc, Wilmington, MA) at the Oxford University Centre for Integrative Neuroimaging (University of Oxford). fMRI data was acquired using a multiband echo-planar imaging (EPI) sequence, with multiband factor 3, TR = 1.235 s, TE = 20 ms, flip angle = 65 degrees, field of view = 216 mm, and a voxel resolution of 2×2×2 mm3. All scans were of axial orientation angled to the long-axis of the hippocampus, covering the whole brain. For each participant, a T1-weighted structural image was acquired to correct for geometric distortion and perform co-registration between EPIs, consisting of 192 1 mm axial slices, in-plane resolution of 1x1 mm2, TR= 1.9 s, TE = 3.97 ms, and field of view = 192 mm. A field map with dual echo-time images was also acquired (TE1 = 4.92 ms, TE2 = 7.38 ms, whole-brain coverage, voxel size 2x2x2 mm3). Physiological measures (cardiac pulse and thoracic movement) were collected during the scan but not used in analysis. Cardiac pulse was recorded using an MRI-compatible pulse oximeter. Thoracic movement was monitored using a custom-made pneumatic belt positioned around the abdomen.

fMRI preprocessing and GLMs
Preprocessing of fMRI data was carried out in SPM12 (https://www.fil.ion.ucl.ac.uk/spm/). The dataset of one participant was excluded due to excessive motion. For the 43 participants included in the fMRI analysis, images were corrected for signal bias, realigned to the first volume of the block, corrected for distortion using fieldmaps, normalised to a standard EPI template and smoothed using a 5 mm full-width at half maximum Gaussian kernel. To remove low frequency noise from the preprocessed data, a high-pass filter was applied to the data using SPM12’s default settings. For each participant and for each scan block presenting bird stimuli, the resulting fMRI data was analysed in an event-related manner using three General Linear Models (GLMs). Explanatory variables used a delta function to indicate the onset of a stimulus and were then convolved with the hemodynamic response function . In addition to the explanatory variables (EVs) of interest (described below), six additional scan-to-scan motion parameters produced during realignment were included in the GLM as nuisance regressors to account for motion-related artefacts in each task block.

The first GLM was used to identify brain regions that represent task stimuli (Supplementary Fig. 5b). 14 EVs were included per scan block. The first ‘repetition suppression’ EV was a parametric regressor corresponding to the number of trials between the current stimulus and the most recent previous presentation of the same stimulus. The regressor was subsequently z-scored. The next 11 EVs accounted for each of the different bird stimuli (bird 1-11). The remaining 2 EVs accounted for detected and undetected ‘oddball’ trials, respectively. All EVs were then convolved with the hemodynamic response function. The contrast of interest was the parametric regressor for repetition suppression, contrasted against baseline.

The second GLM was used to quantify the neural spread of association across the ‘11-node’ underlying task structure. 14 EVs were included per scan block. The first EV was the parametric regressor corresponding to the link distance (nodes) between the presented stimulus and the preceding stimulus measured along the ring structure. The parametric regressor was according to an exponential function (1–2[link distance]) to account for non-linearity in link distance encoding, and was subsequently z-scored. The next 11 EVs accounted for each of the different bird stimuli (bird 1-11). The remaining 2 EVs accounted for detected and undetected ‘oddball’ trials, respectively. All EVs were then convolved with the hemodynamic response function. The contrast of interest was the parametric regressor of link distance (Fig. 5b), contrasted against baseline.

The third GLM was used to quantify the neural spread of association using the output from the ‘6-node’ neural network model. 17 EVs were included per scan block. Similar to the ‘11-node’ GLM described above, the first EV was the parametric regressor corresponding to the link distance between the presented stimulus and the preceding stimulus. An additional parametric regressors was included, which corresponded to the link distance between the presented stimulus and the stimulus that was presented 2 trials back. The two parametric regressors were defined according to the peak firing rate of assemblies in the neural network model (Fig. 4c, middle panel), and subsequently z-scored. As the parametric regressors could only take into account stimuli 1 to 3 links away, 2 additional EVs were included to account for stimuli 4 or 5 links separated on the previous trial and the trial before the previous. As in the 11-node model, the remaining EVs account for the different bird stimuli (11 EVs) and detected and undetected oddball trials (2 EVs). All EVs were then convolved with the hemodynamic response function. The contrast of interest was the main of the two parametric regressors of link distance, contrasted against baseline.

fMRI analysis and statistics
The contrast of interest was entered into second-level random effects ‘group’ analyses to assess the main effect of link distance, and to assess the effect of 4 covariates: GABA+ in LOC, pupil response, and behaviour (‘Overgeneralisation errors’ and ‘Mean rank proximity’). Second-level analyses were performed for each group separately (ATX, PLC), and for all participants combined. We set the cluster-defining threshold to p < 0.01 uncorrected before using whole-brain family wise error (FWE) to correct for multiple comparisons, with the significance level defined as p < 0.05. To assess changes in BOLD in regions of interest where we predicted representation of task stimuli and a spread of association across memory (Fig. 5 c), we performed small volume correction for multiple comparisons, with peak-level FWE correction at p<0.05. For the small volume correction in hippocampus and parahippocampus we use bilateral anatomical masks segmented from several representative T1 images normalised to the MNI152 template (Supplementary Fig. 12a), followed by post hoc tests using unilateral hippocampal or parahippocampal masks. For small volume correction in LOC we used a 10mm radius sphere centered on the group-average MRS voxel (Supplementary Fig. 5a).

To assess the relationship between fMRI cross-stimulus suppression and each of the 4 covariates of interest (GABA+ in LOC, pupil response, ‘Overgeneralisation errors’ and ‘Mean rank proximity’), we ran the second-level random effects model with the inclusion of each covariate at the group level. For the GLM with GABA+, an additional covariate was included to account for differences in the concentration of Glx in LOC. Only participants with reliable MRS model fits were included, resulting in n=19 participants in the ATX group and n=17 participants in the PLC group. To assess the relationship between pupil response and fMRI, a pupil response covariate was defined as the average difference in pupil response to ‘oddball’ stimuli versus regular stimuli, across the time period where we observed a group difference between ATX and PLC (Fig. 3b). Covariates for the concentration of GABA+ and Glx in LOC were included to assess variance predicted by the pupil response, over and above variance explained by changes in GABA+ and Glx. Thus, data for a given participants was only included if pupillometry data quality met the inclusion criteria (defined above) and if MRS data resulted in reliable model fits, resulting in n=15 participants in the ATX group and n=12 participants in the PLC group. To assess the relationship between fMRI cross-stimulus suppression and behaviour, we included a covariate for the behavioural measures of overgeneralisation from the implicit memory test (Fig. 2f,g).

Furthermore, we used a region of interest (ROI) approach to visualise the relationships between fMRI cross-stimulus suppression and GABA+ in LOC, pupil response, and behaviour. To this end, we used anatomical ROIs for right parahippocampus and right hippocampus to extract beta parameter estimates for the parametric link distance regressor, and computed the Spearman rank correlation for each relationship (Fig. 6a-b, e-f; Supplementary Fig. 14).

Recurrent spiking neural network model
All simulations were carried out in C++ using Auryn (http://www.fzenke.net/auryn).
Neuron Model: The postsynaptic neurons were described by point leaky integrate-and-fire neurons with after-hyperpolarisation (AHP) current, whose membrane potential V(t) evolved according to 47:

τm dV(t)/dt = (Vrest - V(t)) + gAHP(t)(VAHP - V(t)) + gAMPA(t)(VAMPA - V(t)) + gGABA(t)(VGABA - V(t)) + gNMDA(t)(VNMDA - V(t)) ( 2 )

where τm = 30 ms is the membrane time constant and Vrest = -65 mV is the resting membrane potential; VAMPA = VNMDA = 0 mV and VGABA = VAHP = -80 mV are the reversal potentials; gAMPA, gNMDA, and gGABA are the synaptic conductances for their respective channels, and gAHP is the conductance of the AHP channel. The neuron fires when its membrane potential crosses a fixed threshold of Vth = -50 mV below, after which it returns to its resting potential where it remains clamped for τEref = 5 ms and τIref = 2.5 ms of refractory period in excitatory and inhibitory neurons, respectively.

Synapse Model: The synapses were conductance-based. The AHP conductance follows the equation 47:

dgAHP(t)/dt = -gAHP(t)/τAHP + A_AHP * Spost(t) ( 3 )

Where τAHP = 100 ms is the characteristic time of the AHP channel, and A_AHP = 5nS is the amplitude of increase in conductance due to a single postsynaptic spike. Spost(t) = ∑ δ(t-tk,post) is the postsynaptic spike train, where tk,post is the time of the kth spike of the postsynaptic neuron, and δ(.) is the Dirac’s delta.

The synaptic conductance for X (where X can be AMPA, GABA, or NMDA) obeys the following equation 47:

dgX(t)/dt = -gX(t)/τX + ∑wj(t)Sj(t) ( 4 )

Here, the characteristic times are τAMPA = 5 ms, τGABA = 10 ms, τNMDA = 150 ms. Sj(t) = ∑ δ(t-tk,j) represents the presynaptic spike train, where tk,j is the time of the kth spike of neuron j.

Plasticity Model: The plasticity model employed in these simulations was the so-called "codependent" plasticity model47 based on multiple empirical findings. Codependent synaptic plasticity rules depend on both spike times and synaptic currents. The codependent plasticity function φ of a synapse is represented as:

φ(E,I,pre,post) ( 5 )

where E and I are the excitatory and inhibitory currents due to neighbourhood synaptic activity (including the synapse's own activity), and pre, post are the spike timings of the pre- and postsynaptic neurons, respectively. E represents the calcium influx through N-methyl D-aspartate (NMDA) channels to model excitatory currents and I represents the chloride influx through gamma-aminobutyric acid (GABA) channels to model inhibitory currents. These currents are defined as follows:

τE dE(t)/dt = -E(t) + ∑INMDA,k(t) ( 6 )

τI dI(t)/dt = -I(t) + ∑IGABA,k(t) ( 7 )

where τE = 10 ms and τI = 100 ms are the characteristic time constants for E and I, respectively. INMDA,k(t) and IGABA,k(t) are the currents flowing through the NMDA and GABA channels for the kth excitatory and inhibitory synapses, respectively.

The learning rule of the jth excitatory synapse onto the postsynaptic neuron is defined as:

dwj(t)/dt = φE(Ej(t),I(t);Sj(t),Spost(t)) = [ALTP xj+(t)Sj(t)Ej(t) - ALTD y-(t)Spost(t)wj(t) - Ahet yE(t)Spost(t)(Ej(t))2]exp [-(I(t)/I*)γ] ( 8 )

where ALTP = 3×10-4 and ALTD = 3×10-5 are the learning rates of Hebbian long-term potentiation and long-term depression, respectively, and Ahet = 1.5×10-8 is the learning rate of the heterosynaptic plasticity, which depends quadratically on the neighbouring excitatory currents. Importantly, in the co-dependent plasticity rules, the heterosynaptic plasticity term counterbalances the destabilising effects of excitatory potentiation. Without the heterosynaptic plasticity term the excitatory synaptic weights do not stabilise and reach a maximum possible value, with excitatory firing attaining implausible firing rates (Supplementary Fig. 9, Supplementary Fig. 10). Heterosynaptic plasticity therefore provides a stabilising mechanism to counteract the excitatory plasticity that enables: (1) embedding of sensory representations (both the PLC and ATX networks); (2) embedding of the associative memories (i.e. ring topology: ATX and PLC); (3) the graded excitatory synaptic weights across assemblies (ATX model only).

I* = 200 and γ = 3 represent the level and shape of the control of inhibitory activity over changes in excitatory weights (Supplementary Fig. 9, Supplementary Fig. 10). Sj(t) and Spost(t) are the pre- and postsynaptic spike trains, as described earlier. xj+(t), representing the trace of the presynaptic spike train, follows:

dxj+(t)/dt = -xj+(t)/τ+ + Sj(t) ( 9 )

y-(t) and yE(t), representing the traces of the postsynaptic spike trains with different time scales, follow:

dy-(t)/dt = -y-(t)/τ- + Spost(t) ( 10 )

and

dyE(t)/dt = -yE(t)/τE + Spost(t) ( 11 )

where τ+ = 16.8ms, τ- = 33.7ms, and τE = 100ms.

The inhibitory learning rule of the jth inhibitory synapse onto the postsynaptic neuron is defined as:

dwj(t)/dt = φI(Ej(t),I(t);Sj(t),Spost(t)) = AISP Ej(t)[Ej(t) - αI(t)][xj(t)Sj(t) + yj(t)Spost(t)] ( 12 )

where AISP = 1×10-3 is the codependent inhibitory plasticity learning rate, α is the excitatory-inhibitory balance point, such that E/I = α. xj(t) and yj(t) are the synaptic traces of the pre- and postsynaptic spike trains, respectively. They follow:

dxj(t)/dt = -xj(t)/τSTDP + Sj(t) ( 13 )

and

dyj(t)/dt = -yj(t)/τSTDP + Spost(t) ( 14 )

where τSTDP = 20ms.

Network and Simulation: In the neural network model, we simulated a recurrent neural network of 1000 excitatory and 250 inhibitory neurons. E-E and I-E connections were plastic, while E-I and I-I connections were static. Connectivity was 5% between and across all neuron groups. Initial weights for the different synapse types were wEE = 0.25 nS, wEI = 0.35 nS, wIE = 0.31 nS, wII = 0.31 nS. Background network activity was maintained by connecting a group of 100 Poisson neurons firing at 1Hz to the excitatory neuron group with a weight of winput = 0.1 nS and connection probability of 1.

Cell Assembly Formation: The cell assemblies 1–6 were formed by connecting six independent Poisson groups (representing external sensory stimuli) of 100 neurons each, firing at 2.5 Hz, to six groups of 100 excitatory neurons each. The synapses between the Poisson neurons and the excitatory neurons had a weight of 0.1±0.01 nS. This resulted in an increase in network excitation, which was subsequently balanced by strengthening wIE (I-E connections) between groups of 25 inhibitory neurons to each of the formed excitatory cell assemblies to wIE = 0.9±0.05 nS. Hence, each assembly 1–6 was composed of 100 excitatory neurons and 25 inhibitory neurons.

Inter-nodal Associations: In both the placebo and atomoxetine neural network simulations, excitatory connections between formed nodes were strengthened by setting E-E connections between them to wEE = 0.43±0.02 nS. However, in the atomoxetine simulations, these E-E connections were strengthened under decreased inhibitory firing, which was achieved by setting wEI to only 97% of its value, or wEI = 0.3395 nS.

In the placebo simulations, this E-E strengthening was followed by setting inter-nodal I-E connections to wIE = 0.9±0.05 nS. In the atomoxetine case, these connections were only strengthened to wIE = 0.7±0.05 nS , which was the minimum I-E strengthening required to prevent runaway excitation due to E-E potentiation triggered by the inter-nodal excitatory associations.

Driving Activity of Assembly ‘1’: To test the degree of coactivation, the activity in assembly ‘1’ was driven externally for 3.3s during the period marked “stimulus” (Fig. 4, centre plots) by increasing the weight of the relevant Poisson group representing the sensory input to 0.5 nS.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript titled ‘Noradrenaline causes a spread of association in the hippocampal cognitive map’ presents a variety of new empirical data that the authors argue, together, indicate that elevated noradrenergic (NE) tone promotes generalization of statistical learning. Human subjects were either administered ATX, an NE-reuptake inhibitor, or a placebo before viewing a sequence of images of two birds and scenes. The combinations of birds and scenes presented across stimuli were constructed according to a pre-defined grammar. Importantly, in this grammar, each bird had only two others it could appear with but, therefore, also had second degree neighbors—birds that were never shown with a particular bird but were shown with the birds that had—enabling implicit and explicit assessments of the associative strength of 2nd degree associations (e.g., via transitive inference). The key observation supporting the main conclusion is shown in Figure 2F, indicating that overgeneralization errors are significantly more likely for those who received ATX than for those who received the placebo. The remainder of the results are used to support the efficacy of the drug in modulating NE levels, modeling of the main result, and evidence of brain activity that aligns with a prediction of the model.\n\nThe described work is elegant and creates a multi-faceted portrait of how modulating NE tone influences neural processing and resulting learning. Overall, I’m impressed at the experimental design and see the pattern of results as being fairly cohesive and moderately convincing. The final story that the authors tell from the data aligns well with the evidence presented. If the result indeed proved reliable, the finding would be impactful and timely. It is of high significance to understand the how modulatory signaling influences the nature of learned representations and the cognitive map is a framework that is having a moment of increased attention. This work addresses both the influence of neuromodulation and a mechanism of cognitive map control. Though I don’t see overt flaws with the work, my enthusiasm remains tempered because of two main issues: 1) minimal data to support primary claims, and 2) little effort to rule out competing hypotheses. These are unpacked below.\n\nMajor issues –\nMinimal data to support primary claims. The entire story that NE increases generalization in the cognitive map is supported by a single empirical comparison (Fig 2F). This comparison appears to have been done rigorously but it is not supported with any secondary analyses of any sort. Given that no other differences were observed between the ATX and placebo conditions, I wonder if how robust this one comparison is. Looking at the raw data points that are plotted in Fig 2F, it seems that only a subset (60%?) of the subjects in the ATX condition exhibited the effect at all. I don’t want to prescribe one versus another way to reaffirm that this is a robust effect, but I’ll say that this one comparison leaves room for concern that the effect may not be robust. Relatedly, for the result about repetition suppression as a function of distance in the ring of stimuli, a significant pattern is observed in one hippocampus for ATX but this was not significantly different from the placebo condition. Though this is something, it isn’t fully convincing. Relatedly, this lack of difference should not be buried in the extended materials.\n\nLittle effort to rule out competing hypotheses. No competing hypotheses were named in the manuscript. Though I’ll agree that the main narrative offered for interpreting the data makes sense, it does not seem to me that it is unique in its ability to account for the data. For example, the modeling presents is one approach to generate spreading associations. Other approaches could also likely work. Likewise, related to the point above, there is little consideration of other possible explanations for the difference in implicit error rate.\n\nMinor issues –\nIt isn’t clear how ‘Overgeneralization errors (%)’ were calculated. The methods indicates that it is the percentage of trials that proximal context is chosen minus the percentage of the trials where any other error was made. Either my understanding of this is wrong or participants are selecting the proximal context even more often than they are selecting the correct context. To unpack – six contexts are shown (16.67% chance), one is correct and this is chosen ~30% of the time (Fig 2), I imagine one of the remaining five is a proximal context (by chance 1 in 5, 20%, of errors should be to a proximal context) while the other four are non-proximal (80% of errors should be this by chance). Fig 2F shows that, even in the for the placebo condition, ‘overgeneralization errors’ are about 0%, which I understand means the proximal context is chosen as often as ‘any other error was made. This means that 50% of the errors, even in the placebo condition are to the proximal context. Given that ~70% of trials were errors, 50% of those would be 35% of all trials – a value higher than the percent of correct trials. I imagine I’m misunderstanding something – hopefully the authors can spot what and clarify as necessary to avoid such distracting misunderstanding.\n\nThe timing of the elevated pupil size is extremely slow relative to the task design. The significant difference is only observed a full trial after an oddball.\n\nFig 1F(top) DABEST representation of data in, using normal distributions, is not well suited to data the data which is not normally distributed.\n\nThe number of participants and/or degrees of freedom should be listed near where stats are reported.", "reviewer_2": "In this study, Koolschijn et al. investigated how noradrenaline (NA) affects forming implicit associations in a learning task in human volunteers. They used the NA reuptake inhibitor atomoxetine in a double-blind placebo-controlled design. Participants performed a smart associative learning task featuring both an explicit and an implicit learning component. While the explicit learning was unaffected by the treatment, atomoxetine increased over-generalization errors in the implicit domain. Physiological markers of increased NA (GABA tone, pupil size) predicted fMRI signs of this over-generalization effect, which in turn predicted behavioral effects. A simple network model could account for the observed ‘spread of associations’. This is an unusually clear model and presentation of a complex and behaviorally relevant concept, with high explanatory power. I only have a few questions and comments.\n\nMajor points\n1. The correlations between physiological signs of drug effect and fMRI signs of ‘spread’, as well as between the fMRI and behavioral signatures suggest that there might be a correlation directly between pupil/GABA tone and over-generalization behavior. Was this tested? If this was feasible to test, it would be interesting to report it.\n2. It would be worthwhile to elaborate on the rationale behind focusing on LOC (is that ‘lateral occipital complex’?). Was a change in GABA tone not expected, as well as observed elsewhere?\n3. The Authors tend to report direct statistical comparisons of the groups, which is the better approach than saying ‘significant in A but not significant in B’. I did not find this for the correlations of the physiological markers and ‘neural spread of associations’ (lines 405-407, 417-419). Was there a significant difference in the direct comparisons? In this regard, the sample sizes are also of relevance; since there were some exclusions according to the Methods, it would make sense to provide all actual sample sizes for the statistical comparisons in the figure legends.\n4. I found the correlation results pertaining to Figure 5 very exciting. At the same time, I was somewhat confused by the figure. What exactly is plotted in the scatter plots in Fig.5E, G and H? Is every dot representing a single participant? If so, why are the plots ‘illustrative’ – is this a selection of the data? If so, it would be better to present all data in the figure.\n5. The Methods state that participants were trained to criterion. Did learning time differ in the two groups? Did learning time show any correlation with the results (behavioral, neural, physiological signs)?\n\nMinor points\n1. Figure legend for panel 1F: subpanel assignments (e.g. upper right) do not seem to fit the image.\n2. ‘Runaway excitation’ suggests an uncontrolled processes: I suggest rephrasing this.\n\nDisclaimer: Not having performed those methods myself, I am not in a good position to judge the methodology for the MRS, and I could only assess the broad approaches with respect to the fMRI methodologies.\n\nI believe that openness and transparency increase the fairness of peer review. Therefore, I am signing my reviews.\nBalazs Hangya", "reviewer_3": "In this paper, the authors studied the effect of noradrenaline on the association of memory items in the formation of the hippocampal cognitive map of human patients. To this end, the authors designed a behavioral task in which the patients learn the association between bird-bird pairs and contextual cues. Presentations of these images had a virtual ring structure, \"- bird - cue - bird - cue -...\" The authors showed that an increasing level of noradrenaline causes the overgeneralization of the bird-bird association over a long distance along the ring. The authors constructed a computational network model to suggest that the overgeneralization is caused by the reduction of inhibitory plasticity by the noradrenergic level increase.\n\nThe experimental paradigm seems to be novel, and the topic is interesting. However, I am unsure whether the results shown here are qualitatively and quantitatively sufficient to warrant publication in Nat Comm. The quantitative information about the overgeneralization is also limited.\n\nMajor concerns:\nSeveral studies have shown that memory engrams encoding sensory and behavioral experiences that occur in a temporal vicinity tend to overlap (e.g., Mitashita et al., Nature 1988; Cai et al., Nature 2016). In addition, several models have been proposed to account for the overlapping cell assemblies (e.g., Griniasty et al., Neural Comput 1993; Haga and Fukai, Phys Rev Lett, 2019; Burns et al., eNeuro 2022). The authors, however, simply assumed that cell assemblies encoding memory items are orthogonal and mutually non-overlapping. This setting of the model looks over-simplified. What experimental data justify this assumption?\n\nRelated to the above comment, there are no quantitative comparisons between the model and activity data (e.g., in Fig. 4), which also limits the validity of the proposed model and inhibitory mechanism of overgeneralization.\n\nIs heterosynaptic plasticity in Eq. 7 crucial for the bird-bird association? How is overgeneralization affected by the heterosynaptic plasticity?\n\nThe explanation of network simulations is confusing. How E-E and I-E connections were modified or fixed in the simulations is hard to understand. For instance, in the Inter-nodai Association, the authors mention w_EE was fixed at the same values, but w_EI was set at different values in the placebo and atomoxetine conditions. Does this imply that the excitatory and inhibitory plasticity rules were not applied to these synapses? Then, which synapses were modifiable?"}, "author_response": "Point-by-point response to Reviewers’ comments\nWe would like to start by thanking the three reviewers for their constructive comments. We\nhave addressed all of your comments in turn below. As you can see, in the revised manuscript\nwe include new analyses and simulations of our computational model, together with substantial\nchanges to the text. We hope you agree that these changes have significantly improved the\nmanuscript. Below we have copied all reviewers’ comments in blue, interleaved with our\nresponses in black.\nReviewer #1 (Remarks to the Author):\n[...]\nWe would like to thank the reviewer for their complimentary remarks. We are grateful to the\nreviewer for highlighting how the study creates a multi-faceted portrait for how noradrenergic\ntone influences neural processing and learning. We agree that there is a pressing need to\nunderstand neuromodulatory signalling at a mechanistic and computational level, and believe\nour findings contribute to this endeavour. We would like to also thank the reviewer for\nconstructive suggestions for improving the manuscript. Below we outline how we have\nimplemented all recommendations.\nMajor issues –\n1. Minimal data to support primary claims. The entire story that NE increases\ngeneralization in the cognitive map is supported by a single empirical comparison (Fig\nNCO MMS-25-23478-T, p. 1\n2F). This comparison appears to have been done rigorously but it is not supported with\nany secondary analyses of any sort. Given that no other differences were observed\nbetween the ATX and placebo conditions, I wonder how robust this one comparison is.\nLooking at the raw data points that are plotted in Fig 2F, it seems that only a subset\n(60%?) of the subjects in the ATX condition exhibited the effect at all. I don’t want to\nprescribe one versus another way to reaffirm that this is a robust effect, but I’ll say that\nthis one comparison leaves room for concern that the effect may not be robust.\nThank you for this important point. In the revised manuscript we now provide secondary\nanalyses to demonstrate that the effect shown in Figure 2F is robust to alternative mathematical\ndefinitions of “overgeneralisation error”.\n[...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revisions and responses to all my comments were thoughtful and thorough. I have no further concerns of substance. I wish the authors and their manuscript continued success.", "reviewer_2": "The Authors were responsive to my points. I think this is an exciting study and I have no further concerns.\n\nI believe that openness and transparency increase the fairness of peer review. Therefore, I am signing my reviews.\nBalazs Hangya", "reviewer_3": "I appreciate that the authors have revised their manuscript extensively in response to my comments. The novel simulation results and modifications made in the revised manuscript clarified all my concerns. I have no further comments on the manuscript. I feel this manuscript presents an interesting piece of work."}, "author_response": ""}]

Decision letter