Most memories that are formed are forgotten, while others are retained longer and are subject to memory stabilization. We show that non-invasive transcutaneous electrical stimulation of the greater occipital nerve (NITESGON) using direct current during learning elicited a long-term memory effect. However, it did not trigger an immediate effect on learning. A neurobiological model of long-term memory proposes a mechanism by which memories that are initially unstable can be strengthened through subsequent novel experiences. In a series of studies, we demonstrate NITESGON’s capability to boost the retention of memories when applied shortly before, during, or shortly after the time of learning by enhancing memory consolidation via activation and communication in and between the locus coeruleus pathway and hippocampus by plausibly modulating dopaminergic input. These findings may have a significant impact for neurocognitive disorders that inhibit memory consolidation such as Alzheimer’s disease. Research on enhancing and preserving human memory has substantially increased in the last few decades due largely to the prevalence and inexorable condition of Alzheimer’s disease (AD). Early behavioral indicators of AD include a decline in an individual’s ability to retain learned information and remember events, situations, and objects, alluding to synaptic connections losing strength as AD emerges . As a reThere is an ongoing debate about whether non-invasive electrical stimulation of the scalp modulates the excitability of neurons directly . InteresMost episodic-like memories that are formed are forgotten, while others are retained for longer periods of time and are subject to memory stabilization . This isThe present study tests the above hypothesis questioning if NITESGON induces a long-term memory effect by strengthening memories via behavioral tagging across eight experiments. The first set of experiments aims to confirm the behavioral tagging hypothesis as the potential mechanism inducing memory consolidation via NITESGON, whereby the second set of experiments examines the underlying brain network involved in synaptic consolidation and investigates the underlying neural mechanism that is associated with behavioral tagging induced by NITESGON.The idea behind behavioral tagging suggests that weak memories that are regularly unstable and likely to be forgotten will solidify following a novel experience . That isSeeing that NITESGON activates the LC pathway, which plays an important role in memory consolidation, we hypothesize that participants will be able to establish long-term memories upon modulating the LC both during learning, as shown before , as wellTo further explore the effect of NITESGON, resting-state EEG (rsEEG) and salivary α-amylase (sAA) were collected immediately before and after NITESGON on visit 1. Previous research has revealed an increase in sAA, a putative marker of endogenous NA activity, immediately following NITESGON . FurtherF = 0.24, p = 0.79; see F = 5.24, p = 0.009; see F = 7.69, p = 0.010; see r = 0.59, p < 0.001; rs = 0.62, p < 0.001; r = 0.47, p = 0.09 and after learning ) between memory recall 7 days later and sAA levels on visit 1 (pre vs. post). No significant correlation was obtained for the sham group . Memory recollection 7 days after stimulation was associated with increased gamma power in the medial temporal cortex as well as the precuneus and dorsal lateral prefrontal cortex immediately after stimulation  .Prior research on behavioral tagging has shown that items paired with an electric shock  had a retroactive memory effect on items learned before the fear conditioning task . This prTo test the hypothesis, Experiment 2 had participants take part in a word-association task followed by a spatial-navigation object-location task while receiving active or sham NITESGON during the second task. These two types of tasks were selected because they would not interfere with one another, seeing that both require different episodic information. rsEEG data and sAA were collected immediately before and after the two tasks on visit 1. Two memory tests were taken 7 days after learning the word-association and spatial-navigation tasks.F = 0.32, p = 0.73; see F = 0.09, p = 0.98) and second  tasks between the active and sham NITESGON groups. On visit 2, 7 days after initial learning, a significant effect was obtained for recall  and second tasks , revealing an increase in word recall (46.26 ± 3.76% vs. 37.88 ± 9.88%), as well as object-location recall (51.82 ± 7.75% vs. 44.39 ± 3.68%) for the active group in comparison to the sham group. Furthermore, a significant increase in sAA  between memory recall 7 days later and sAA levels on visit 1 (pre vs. post). For the sham group a significant effect was obtained between memory recall 7 days later and sAA levels on visit 1 (pre vs. post) for the first task . No significant correlation was obtained for the sham group for the second task . Memory recollection 7 days after stimulation was associated with increased gamma power in the medial temporal cortex immediately after stimulation for both the first  and the second task  . During the second visit, 7 days after learning the tasks, participants that received active NITESGON  and the second task   than those who received sham NITESGON, indicating that the active NITESGON group (44.27 ± 10.97) showed significant improvement in comparison to the sham NITESGON group (30.46 ± 15.13) for the word-association task. For the object-location task, the active NITESGON group (53.33 ± 11.29) demonstrated a significant increase in the number of correctly recalled objects-locations than the sham NITESGON group (44.17 ± 7.75). Our data revealed that there was a significant interaction effect for sAA  immediately after learning. No significant difference  was obtained in sAA for the active group (78.72 ± 47.67) in comparison to the sham group (76.77 ± 39.87) before learning the association tasks. This increase in sAA seen in the active group correlated with how many items they recalled 7 days after the learning phase for both the word-association task  and sham groups  between memory recall 7 days later and sAA levels on visit 1 (pre vs. post). Memory recollection 7 days after stimulation was associated with increased gamma power in the medial temporal cortex immediately after stimulation for both the first  while receiving either active or sham NITESGON during the first task. rsEEG data and sAA were collected immediately before and immediately after the two tasks on visit 1. Two memory tests were taken 7 days after learning both word-association tasks.F = 0.84, p = 0.37; see F = 0.27, p = 0.61) and second tasks  between the active and sham NITESGON groups. Seven days later, during the recall phase, we found a significant interaction effect for recall  and the second tasks , a significant increase in number of words correctly recalled was observed in the active  compared to the sham group .On visit 1, no significant difference  in comparison to the sham (13.00 ± 6.65%), indicating that the interference effect plays less of a role in the active group.Upon assessment for a potential interference effect, the active group displayed a significant difference in how many words participants were able to recall between the first and the second tasks (18.31 ± 7.03%)  immediately after the learning. However, no significant difference  was obtained in sAA for the active group (78.72 ± 47.67) in comparison to the sham group (76.77 ± 39.87) before learning the word-association tasks. This increase in sAA correlates with how many words they recalled 7 days after the learning phase for both the first word-association task  or sham group  between memory recall 7 days later and sAA levels on visit 1 (pre vs. post) for the first task. For the second task, we found a significant correlation for both the active  and sham groups  was obtained between memory recall 7 days later and sAA levels on visit 1 (pre vs. post) for the first task.Our data revealed that there was a significant interaction effect for sAA . Both brain areas enhance retention of everyday memories in the hippocampus . MoreoveWe conducted a resting-state functional connectivity magnetic resonance imaging study to verify the relationship between changes in the LC and hippocampus as well as the VTA and hippocampus. We hypothesized that participants who received active NITESGON would show increased activity in the LC and hippocampus, but not in the VTA, and increased functional connectivity between the LC and hippocampus, but not between the VTA and hippocampus. We scanned in three consecutive blocks: immediately before, during, and immediately after stimulation. NITESGON was applied at a constant current of 1.5 mA for 20 min via electrodes placed over the left and right C2 nerve dermatome.F = 4.34, p = 0.023), VTA  and hippocampus  when comparing the active and control groups  and after  stimulation for the active group in comparison to the sham group. Before stimulation, no significant difference was obtained between the active and sham groups . For the VTA, a significant increase was obtained during  stimulation for the active group in comparison to the sham group. Before  or after  stimulation, no significant difference was obtained between the active and sham groups. To further confirm our data, we replicated our analysis by not including a smoothing kernel, showing similar results for the LC, VTA, and hippocampus. Furthermore, we included two control areas, the left inferior parietal cortex, where we do not expect to see any changes  was inspected to verify if NITESGON evoked activity changes in the LC, VTA, and hippocampus. Our findings showed a significant effect for the LC , but not between the right hippocampus and VTA   and after stimulation . However, no significant effect was obtained between the LC and right hippocampus for the active group relative to the sham group before stimulation .Furthermore, a regions of interest (ROI)-to-ROI analysis demonstrated an effect between the right hippocampus and LC (.76) see . AdditioThe previous experiment revealed activity changes in both the LC and hippocampus as well as increased connectivity between the LC and hippocampus both during and after NITESGON. Conversely, the VTA did not show changes in activity after stimulation or connectivity changes between the VTA and hippocampus during or after stimulation. However, activity changes in the VTA during NITEGSON were detected. Previous animal research has identified selective neuronal connections between the LC and VTA, implying an interaction between the LC and VTA during NITESGON may exist .A key neuromodulator in memory consolidation is dopamine (DA) . DA affeTo regulate synaptic plasticity, the hippocampus receives dopaminergic input from the VTA and the LC . HoweverA proxy for DA is spontaneous eye blink rate (sEBR), or the frequency of blinks per unit of time . PharmacsAA, pupil size as well as neurophysiology  are common proxies for LC–NA activity. More specifically, pupil diameter indexes LC activity in both monkeys and humans , findingF = 11.61, p = 0.003; see F = 9.10, p = 0.007) after NITESGON. Before NITESGON no significant difference  was observed in the active group (26.80 ± 8.24) relative to the sham group (20.45 ± 12.63) in sEBR. Also, a significant interaction effect for sAA by condition  after stimulation for the active group (132.82 ± 51.23) in comparison to the sham group (81.22 ± 45.43). No significant difference  was obtained when comparing the active group (72.42 ± 43.77) versus the sham group (70.10 ± 19.93) before stimulation. Peak-to-peak amplitude analysis for P3 electrode further showed a significant effect . In addition, a significant effect was shown for active NITESGON after stimulation in comparison to before stimulation . A positive correlation was obtained between the difference in sEBR, and sAA  in comparison to sham (−3.59 ± 4.80) stimulation . Our findings demonstrated that participants could not accurately determine if they were assigned to the active or sham NITESGON group in each experiment, suggesting that our sham protocol is reliable and well blinded see .Taken together, our experiments support the hypothesis that NITESGON targeting the LC strengthens hippocampal memories via the behavioral tagging mechanism. NITESGON increases activity in the LC and hippocampus during and immediately after stimulation and increases connectivity between these two areas, thus instigating initial memory consolidation and increasing the retention of memories that are formed within a window of opportunity spanning before and after LC activation. This is in accordance with the construct of behavioral tagging, which explains how memories that would normally be forgotten will endure in memory preceding, during, and following activation of the LC pathway .Notably, NITESGON does not generate an immediate memory effect during learning, however, a favorable behavioral effect is seen 7 days after initial learning. Although previous research has suggested that millisecond pairing between nerve stimulation and auditory or motor learning is essential to induce targeted plasticity , our datPrevious research demonstrated that LC discharge enhances synchronization of gamma activity in the hippocampus in rats  and thatThe role of the LC–NA system in synaptic plasticity and molecular memory consolidation has been well established over the past decades . HoweverA prevailing hypothesis is that hippocampus-dependent memory is mediated by a subiculum–accumbens–pallidum–VTA pathway via DA . Our resAlthough our results reflect the putative tag-and-capture mechanism, several emotional arousal and/or noradrenergic-related frameworks may explain advantages during the consolidation phase that lead to memory enhancement effects that were observed ; therefoWhen interpreting the current findings, it must be considered that some limitations exist within the research; limitations on experimental design are noted below, followed by a discussion of utilizing indirect proxy measures. The task order for Experiment 4 was randomized during the first visit for training and the recall-only memory test 7 days later; however, the word-association and spatial-navigation task used in Experiments 2 and 3 were not counterbalanced; therefore, the findings of Experiments 2 and 3 could have been impacted by a potential order effect. Additionally, in Experiment 5, the learning and test phases for the two groups were conducted at different times of day , thus introducing the potential for circadian effects between the two groups. Furthermore, the recall-only memory testing occurred at the 12 hr point rather than 7 days later, allowing us to conclude that the observed effect seen 12 hr later was not affected by sleep; however, it remains unclear whether the 7-day long-term memory effects of NITESGON are sleep dependent. Moreover, it must also be acknowledged that Experiments 5 and 8 did not include a control-sham stimulation group, thus limiting the interpretation of these two experimental findings. Control-sham stimulation groups would increase our confidence in our findings that NITESGON’s memory-enhancing effects depend not on sleep but on DA receptor activity.Although the use of indirect proxy measures, such as sAA for NA activity and sEBR for DA activity, enabled the tracking of LC–NA activity changes from baseline measurements and demonstrated the potential of an LC–DA relationship, caution must be advised when interpreting results considering these proxy measures are affiliated with limitations, such as being substantially variable, as well as the potential of other brain regions and monoamine neurotransmitters being associated with changes seen in sAA concentration levels , an enzyIn conclusion, our work provides evidence that NITESGON is involved in the consolidation of information rather than encoding. Our findings support an implication previously put forward in the formulation of the synaptic tag-and-capture mechanism proposing late-phase LTP of synaptic activity could explain enhanced memories. As deficits in episodic memory specifically related to memory consolidation are one of the earliest detectable cognitive abnormalities in amnestic mild cognitive impairment and AD , NITESGOAll experiments were designed as a prospective, double-blinded, placebo-controlled, randomized parallel-group study where the researcher and the participant were blinded to the stimulation conditions. Experiment 6 was alone a single-blinded study where the participant was blinded to the stimulation condition, but not the researcher. All experiments were in accordance with the ethical standards of the Declaration of Helsinki. Experiments 1–7 were approved by the Institutional Review Board at the University of Texas at Dallas . All participants signed a written informed consent and consent to publish was obtained.http://www.neuroconn.de). For each participant receiving NITESGON, the anodal electrode was placed over the left C2 nerve dermatome and the cathodal electrode was placed over the right C2 dermatome. To maintain consistency across all participants, research assistants were trained to map out the placement according to the length of the participant’s head.In all experiments, direct current was transmitted via a saline-soaked  pair of synthetic sponges (5 × 7 cm) and was delivered by a specially developed, battery-driven, constant current stimulator with a maximum output of 10 mA  until it reached its programmed maximum output (1.5 mA). After stimulating for the desired duration per group (active or sham), the current was ramped-down  denoting the end of the stimulation. The impedance under each electrode was maintained under 10 kΩ. The ramp-up, ramp-down, and stimulation times were different depending on condition (active vs. sham) and experimental needs.Participants were 48 healthy, right-handed, native-English speaking adults  with a similar educational background  with normal to corrected vision, who all had the maximum score on the Mini Mental State Examination. Participants were screened  prior to enrolling into the study. None of the participants had a history of major psychiatric or neurological disorders, or any tES contraindications, including previous history of brain injuries or epileptic insults, cardiovascular abnormalities, implanted devices, taking neuropsychiatric medications, prescribed stimulants use, or chronic use of illicit drugs .Participants were excluded from the study if screening discovered they were familiar with Swahili/Arabic language or Swahili culture due to the nature of the stimuli. Furthermore, participants received instructions advising them to abstain from the following products for the associated time window prior to their study session: dental work for 48 hr, alcohol for 24 hr, caffeine and nicotine for 16 hr, and hair styling products the day of. Participants provided written, informed consent on the day of the study session.N paradigm , participants were instructed to read and remember 75-sequentially presented Swahili–English  word pairs made up of common day-to-day words. The Swahili–English word pairs were taken from the study by Associative memory performance was measured using a computerized Swahili–English verbal paired-associative learning task. This task was adapted from a well-established study design published in Science by 2) during each of the 4 study blocks, resulting in a total stimulation time of 25 min  and ramp-down time of 30 s. For the sham NITESGON group, the current intensity was ramped-up to 1.5 mA over 30 s and immediately ramped-down over 30 s. Hence, sham NITESGON only lasted 60 s per study period, resulting in a total time of 240 s (60 s × 4 blocks) of stimulation when delivered during the study phase. For the group that received active NITESGON after the word-association task, this consisted of 30 s ramp-up and ramp-down time with 25 min of constant current stimulation at 1.5 mA. The sham NITESGON delivered after the word-association task only consisted of 30 s of ramp-up and ramp-down time resulting in 60 s of stimulation. The rationale behind the sham procedure was to mimic the transient skin sensation at the beginning of active NITESGON without producing any conditioning effects on the brain.There were three groups – active NITESGON during learning  and sham NITESGON immediately after the word-association task; sham NITESGON during learning and active NITESGON immediately the word-association task in the memory consolidation period and sham NITESGON both during and after learning of the word-association task – with 16 participants each. Active NITESGON consisted of ramp-up time of 30 s followed by a constant current of 1.5 mA (current density 0.4285 A/mhttp://compumedicsneuroscan.com). The impedance on each electrode was maintained at less than 5 kΩ. The data were sampled using the Neuroscan Synamps2 amplifier at 500 Hz with online band-pass filtering at 0.1–100 Hz.Continuous EEG data were collected from each participant pre- and post-NITESGON procedures. The data were collected using a 64-channel Neuroscan Synamps2 Quick Cap configured per the International 10–20 placement system with the midline reference located at the vertex and the ground electrode located at AFZ using the Neuroscan Scan 4.5 software  were obtained in a dark room which was dimly lit with a small lamp with each participant sitting upright in a comfortable chair; data collection lasted approximately 5 min. Participants were instructed not to drink alcohol 24 hr prior to EEG recording or caffeinated beverages 1 hr before recording to avoid alcohol- or caffeine-induced changes in the EEG stream. The alertness of participants was checked by monitoring both slowing of the alpha rhythm and appearance of spindles in the EEG stream to prevent possible enhancement of the theta power due to drowsiness during recording . No parthttps://salimetrics.com). The participants were requested to collect 2 ml of saliva in one straight flow and avoid breaks between drool as much as possible. The length of time to collect 2 ml of saliva was noted and the timer was started only when participants began to passively drool into the tube. All saliva samples were stored in 2 ml cryovials, and immediately stored in an −80°C laboratory freezer. Prior to saliva collections, all participants were instructed to avoid food, sugary drinks, excess caffeine, nicotine, and acidic drinks for at least 1 hr before collecting the saliva samples. Participants were also instructed to avoid alcohol and vigorous exercise 24 hr prior to and avoid dental work 48 hr prior to their appointment. In addition, participants were instructed not to brush their teeth within 45 min of sample collection in order to avoid any risk of lowering pH levels and influencing bacterial growth. If the study was scheduled for the afternoon, participants were requested to avoid taking naps during the day. Upon completion of the collection procedures, all saliva samples were packed in dry ice and sent to the Salimetrics laboratory for analysis. We chose to use sAA as a biomarker for norepinephrine as it provided a non-invasive yet valid indicator of central sympathetic nervous system activation  effects on associative memory performance, but did not receive NITESGON, nor were they able to review word pairs. A third researcher who was not responsible for the task or NITESGON conducted the second visit.For the EEG preprocessing, the data were resampled to 128 Hz, band-pass filtered (Finite Impulse Response filter) to 2–44 Hz, and re-referenced to the average reference using EEGLAB 14_1_1b . The EEG2) without assuming a predefined number of active sources. The sLORETA solution space consists of 6239 voxels (voxel size: 5 × 5 × 5 mm) and is restricted to cortical gray matter and hippocampi, as defined by the digitized Montreal Neurological Institute (MNI) 152 template (Standardized low-resolution brain electromagnetic tomography (sLORETA) was used to estimate the intracerebral electrical sources that generated the scalp-recorded activity in each of the gamma frequency bands (30.5–44 Hz) . sLORETAtemplate . Scalpbttemplate .The tomography of sLORETA has received considerable validation from studies combining sLORETA with other more established spatial localization methods such as fMRI , structuFor visit 1 learning, a one-way analysis of variance (ANOVA) was conducted with the cumulative learning rate over the different study periods as the dependent variable and three groups as the between-subjects variable. To look at the memory effect  7 days after learning, we applied a one-way ANOVA with the group as the between-subjects variable and correctly recalled words as the dependent variable.Using the saliva collected via the passive drool method, sAA levels were measured. We conducted a repeated measures ANOVA with groups as between-subjects variable and sAA as within-subjects variable. A simple contrast analysis was applied to compare the different conditions using a Bonferroni correction.t-statistic for paired groups and corrected for multiple comparisons (p < 0.05). The significance threshold for all tests was based on a permutation test with 5000 permutations and corrected for multiple comparisons  were performed for each contrast employing a parisons .Participants were 20 healthy, right-handed, native-English speaking adults  with a similar educational background . Participants were screened and enrolled similar to Experiment 1.Associative memory performance was measured using the same computerized Swahili–English verbal paired-associative learning task used in Experiment 1, however, the task consisted of three study periods in which participants were asked to read and remember 50 Swahili–English word pairs in each study period (50 × 5 = 250 s).N paradigm, participants were instructed to view and remember 50-sequentially presented objects locations on a blue–red–gray background grid with an eye-to-screen distance of ~24 inches across three study-test blocks. The objects consisted of black and white line drawings from the Boston Naming Test; 10 objects from each of the following categories were used: animals, foods, modes of transportation, tools, and household objects. Each image appeared in a randomized order at a randomized location. Objects were presented one at a time for 5 s each . Objects were presented within a white-box background (4.88 × 4.88 cm) and had a red dot superimposed at the object center to mark the precise location. Participants were instructed to study and remember the object-locations as accurately and precisely as possible. After each study phase, a cued-recall test was administered. During the test period, the studied objects were presented one at a time in the center of the screen (in a randomized order), and participants were required to recall the studied locations. At the beginning of every trial, a 2-s fixation cross at the center of the screen was presented. After this 2-s period, participants were able to use the mouse to move the object from the center of the screen to its recalled location and click a button on the mouse to indicate its final location.Participants partook in a second memory performance task immediately after the word-association task. The second memory task consisted of a spatial-navigation object-location association task that was based on previous research . Using tThe Swahili–English verbal associative task was used as task 1 and the spatial-navigation object-location task was used as task 2 for all participants.All participants received sham NITESGON during each study period of task 1 using the following parameters: a 5-s ramp-up period, followed by a constant current of 1.5 mA for 15 s, ending with a ramp-down period of 5 s, allowing for an emulated sensation of the active NITESGON. For task 2, 10 participants received active NITESGON and 10 participants received sham NITESGON. Sham stimulation parameters were the same as used in task 1 and stayed consistent in each of the three study periods of task 2. Participants given active NITESGON received a 5-s ramp-up period, followed by a constant current of 1.5 mA for 250 s, and finished with a 5-s ramp-down period during each of the three study periods of task 2 on the first day. Thus, the total simulation time for the active group was 750 s  and the sham group was 45 s . Just before the first study period of the first task participants NITESGON was delivered for 15 s to help participants habituate to the sensation and to check if they were comfortable with the sensation.Continuous EEG data were collected from each participant pre- and post-NITESGON procedures as detailed in Experiment 1.Saliva was collected twice during each experiment: once immediately prior to NITESGON stimulation and once immediately after NITESGON stimulation as detailed in Experiment 1.Eligible participants were scheduled for two visits to complete the study. Visit 1 consisted of the word-association task  paired with sham stimulation, and then were randomly assigned to either the active or sham NITESGON condition for the spatial-navigation task . The researcher who controlled the NITESGON device was not involved in instructing the participant; this was performed by a second researcher who was blind to the stimulation protocol. rsEEG and saliva were collected twice for all participants, once immediately before and once immediately after NITESGON application. Participants were asked to refrain from studying or searching for the learned word pairs throughout the week. Participants returned 7 days after their first visit for memory testing on both tasks 1 and 2 to measure possible (long-term) effects on associative memory performance, but did not receive NITESGON, nor were they able to review word pairs or objects’ locations. A third researcher who was not responsible for the task or NITESGON conducted the second visit.The continuous EEG data were preprocessed and the source-level gamma activity pre- and post-NITESGON procedures for the two groups was determined as detailed in Experiment 1.For visit 1 learning, a multivariate analysis of variance (MANOVA) was conducted with the cumulative learning rate over the different study periods for both tasks as the dependent variable and group as the between-subjects variable. To look at the memory effect  7 days after learning, we applied an MANOVA with groups as the between-subjects variable and correctly recalled words on both tasks as the dependent variable. For both analyses, if significant, two separate one-way ANOVAs were applied with groups as the between-subjects variable and correctly recalled words as dependent variable for task 1 or 2, respectively.Using the saliva collected via the passive drool method, sAA levels were measured which were compared between the groups as detailed in Experiment 1.A whole brain analysis was used to compare gamma activity before and after NITESGON. This activity was correlated with the number of correctly recalled items (words/locations) 7 days later as detailed in Experiment 1.Participants were 24 healthy, right-handed, native-English speaking adults  with a similar educational background . Participants were screened and enrolled similar to Experiment 1.Associative memory performance was measured using the same computerized Swahili–English verbal paired-associative learning task used in Experiment 2.Participants partook in a second memory performance task immediately after the word-association task consisting of a spatial-navigation object-location association task used in Experiment 2.The same device, electrode placement, and active and sham NITESGON parameters described in Experiment 2 were used. Differing from Experiment 2, Experiment 3 had all participants receive active NITESGON during each study period of task 1 as opposed to task 2. Twelve participants received active NITESGON and 12 participants received sham NITESGON during the first task.Continuous EEG data were collected from each participant pre- and post-NITESGON procedures as detailed in Experiment 1.Saliva was collected twice during each experiment: once immediately prior to NITESGON stimulation and once immediately after NITESGON stimulation as detailed in Experiment 1.Eligible participants were scheduled for two visits to complete the study. Visit 1 consisted of the word-association task  paired with either active or sham NITESGON, and spatial-navigation task  paired with sham NITESGON. The researcher who controlled the NITESGON device was not involved in instructing the participant; this was performed by a second researcher who was blind to the stimulation protocol. rsEEG and saliva were collected twice for all participants, once immediately before and once immediately after NITESGON application. Participants were asked to refrain from studying or searching for the learned word pairs throughout the week. Participants returned 7 days after their first visit for memory testing on both tasks 1 and 2 to measure possible (long-term) effects on associative memory performance, but did not receive NITESGON, nor were they able to review word pairs or objects’ locations. A third researcher who was not responsible for the task or NITESGON conducted the second visit.The continuous EEG data were preprocessed and the source-level gamma activity pre- and post-NITESGON procedures for the two groups was determined as detailed in Experiment 1.The learning in visit 1 and memory performance in visit 2 was compared between the groups as detailed in Experiment 2.Using the saliva collected via the passive drool method, sAA levels were measured which were compared between the groups as detailed in Experiment 1.A whole brain analysis was used to compare gamma activity before and after NITESGON. This activity was correlated with the number of correctly recalled items (words/locations) 7 days later as detailed in Experiment 1.Participants were 32 healthy, right-handed, native-English speaking adults  with a similar educational background . Participants were screened and enrolled similar to Experiment 1. Experiment 4 added familiarity of Japanese language or culture to the participant screening procedure, if indicated, the participant was excluded from the study due to the nature of the stimuli.Associative memory performance was measured using two computerized verbal paired-associate learning tasks. Similar to Experiments 2 and 3, one task comprised of the Swahili–English vocabulary learning, and the second task consisted of a newly introduced Japanese–English  word-association task. The Japanese–English word-association task used the same Swahili–English word pairs, however, the Swahili words were replaced by Japanese words.The same device, electrode placement, and active and sham NITESGON parameters described in Experiment 2 was used. Sixteen participants received active NITESGON and 16 participants received sham NITESGON during the first task, where everyone received sham NITESGON during the second task.Continuous EEG data were collected from each participant pre- and post-NITESGON procedures as detailed in Experiment 1.Saliva was collected twice during each experiment: once immediately prior to NITESGON stimulation and once immediately after NITESGON stimulation as detailed in Experiment 1.Eligible participants were scheduled for two visits to complete the study. Visit 1 consisted of two word-association tasks, whereby task 1was paired with either active or sham NITESGON followed by a second word-association task  paired with sham NITESGON. The order of the two word-association tasks was randomized over the participants in a 1:1 ratio to remove a possible order effect. The researcher who controlled the NITESGON device was not involved in instructing the participant; this was performed by a second researcher who was blind to the stimulation protocol. rsEEG and saliva were collected twice for all participants, once immediately before and once immediately after NITESGON application. Participants were asked to refrain from studying or searching for the learned word pairs throughout the week. Participants returned 7 days after their first visit for memory testing on both tasks 1 and 2 to measure possible (long-term) effects on associative memory performance, but did not receive NITEGSON, nor were they able to review word pairs. A third researcher who was not responsible for the task or NITESGON conducted the second visit. As during the first visit, the two word-association tasks were randomized over the participants in a 1:1 ratio to remove a possible order effect.The continuous EEG data were preprocessed and the source-level gamma activity pre- and post-NITESGON procedures for the two groups was determined as detailed in Experiment 1.For visit 1 learning, a repeated measures ANOVA was applied with the cumulative learning rate over the different study periods for both tasks as within-subjects variable and group (active vs. sham) as between-subjects variable. A similar analysis was applied for the memory effect  7 days after learning. A simple contrast analysis was applied to compare the difference conditions using a Bonferroni correction. In addition, an interference effect was determined by calculating how many words recalled during memory task 2 had an overlap from memory task 1. The higher percentage the more overlap in remembering the English translation for both exact word in tasks 1 and 2, the lesser the interference effect. A one-way ANOVA was applied with the interference effect as dependent variable and group (active vs. sham) as between-subjects variable.Using the saliva collected via the passive drool method, sAA levels were measured which were compared between the groups as detailed in Experiment 1.A whole brain analysis was used to compare gamma activity before and after NITESGON. This activity was correlated with the number of correctly recalled items (words/locations) 7 days later as detailed in Experiment 1.Participants were 20 healthy, right-handed, native-English speaking adults  with a similar educational background . Participants were screened and enrolled similar to Experiment 1.Associative memory performance was measured using the same computerized Swahili–English verbal paired-associative learning task used in Experiment 1.All participants received active NITESGON during each of the four study periods on visit 1 using the following parameters: a 5-s ramp-up period, followed by a constant current of 1.5 mA for 375 s (75-word pairs × 5 s), and finished with a 5-s ramp-down period. The total stimulation time was 25 min . Before the start of the first study period, an additional 15-s habituation period was added to make sure the participants got used to the sensation.Eligible participants were scheduled for two visits to complete the study. Visit 1 consisted of the word-association task paired with active NITESGON. Participants were randomly assigned to one of the two groups (sleep vs. no sleep).As Experiment 4 reveals that NITESGON is involved in synaptic consolidation that occurs over a timespan of minutes to hours after encoding the information, where sleep that has been association with systems consolidation does not play a central role. To cross validate these findings, we opted for the parallel design not including a control condition, as the aim of this study is to see if sleep mediates the memory effect induced by NITESGON, rather than the effect of NITESGON or the interaction between sleep and NITESGON on memory.Ten participants learned the word-association task at 8:00 a.m. and were tested the same day at 8:00 p.m., while the other 10 participants learned the word-association task at 8:00 p.m. and were tested the next day at 8:00 a.m. after a night of sleep. Participants were asked to refrain from studying or searching for the learned word pairs for at least the next 12 hr. The researcher who controlled the NITESGON device was not involved in instructing the participant; this was performed by a second researcher who was blind to the stimulation protocol. A third researcher who was not responsible for the task or NITESGON conducted the second visit (12 hr later).A one-way ANOVA with group (sleep vs. no sleep) as between-subjects variable and number of words correctly recalled as dependent variable was performed.Participants were 32 healthy, right-handed, native-English speaking adults  with a similar educational background . Participants were screened and enrolled similar to Experiment 1. Experiment 6 added the exclusion of those participants who had any contraindication for MRI .3. During scanning, foam padding and earplugs were used to minimize the head movement and scanner noise. An MR-compatible, battery powered NITESGON system manufactured by MR NeuroConn Co (Germany) was applied to each participant inside the MR scanner. All the operating parts and devices that go into the scanner room were MR compatible and everything else was in the control room, connected via the waveguide. The NITESGON system was fully charged before each session and its impedance level was measured regularly to test if it was maintained at approximately 5 kΩ on each end .The resting-state fMRI data were collected on a 3T MR scanner  using a 32-channel SENSE phased-array head coil. The dimensions of the coil was 38 (height) × 46 (width) × 59 (length) cmThe MR session with NITESGON was divided into three consecutive blocks of scanning: before stimulation, during stimulation, and after stimulation. At the beginning of the pre-stimulation session, routine survey and T1 anatomical images were acquired for a total time of 5 min. Before acquiring the T1 image, saline-soaked NITESGON electrodes were positioned on the subject for three consecutive blocks of rsfMRI. For each of the scanning blocks, we acquired 20-min long rsfMRI images.2, at a 1 × 1 × 1 mm3 resolution.For the T1 (MPRAGE) anatomical scan, parameters included a repetition time (TR) of 2300 ms, an echo time (TE) of 2.94 ms, an inversion time (TI) of 900 ms, and a flip angle of 9°. One hundred and sixty sagittal slices were taken, using a matrix size of 256 × 256 mm2, matrix  = 64 × 64, number of slices  = 53 with voxel size = 3 × 3 × 4 mm3 with no gap between slices. The total number of acquired volumes was 400, counting for 20 min.Resting-state fMRI sequences were acquired with the following imaging parameters (echo-planar imaging protocol): TR/TE = 3000/30 ms, field-of-view  = 220 × 220 mmz-direction slices acquired in an interleaved manner. After co-registration of functional volumes to the structural image (T1), volumes that contain extreme movements were linearly regressed out as covariates using Artifact Detection Tool . Functional images were normalized to the standard template using nonlinear transformation parameters acquired by the process of normalizing structural image to the standard template. The normalized functional images were smoothed using 6 mm Gaussian kernel.MR images were preprocessed using Statistical Parametric Mapping . Images from the first five TRs in the beginning of each session were discarded. We normalized high-resolution structural images to a standard MNI template, and segmented for three structural components, which were gray matter, white matter, and cerebrospinal fluid. Functional images were realigned to the middle volume to correct for motion artifacts. Slice-timing correction was adjusted for temporal discrepancies between After preprocessing, the images were processed to account for motion-related and physiological noises using an independent component analysis. Confounding factors of signals from white matter and cerebrospinal fluid were linearly regressed out from the global average brain signal using CompCor. Global signal regression was performed using the grand averaged signal from the gray matter volume. When removing confounding effect, the signal was simultaneously filtered from 0.01 to 0.17 Hz, where the maximum detectable frequency of the signal is 0.167 Hz (TR = 3 s).Shielded cables connected the MR-compatible box and electrodes, and the stimulation data were transferred via the CAT.6 LAN cable that runs throughout the MR scanner room to the non-MR-compatible stimulation devices in the control room. For the active NITESGON group, the current was ramped-up for 30 s followed by a constant current of 1.5 mA for 20 min and a 10-s ramp-down period. For sham NITESGON, the current was ramped-up over 30 s to reach the intensity of 1.5 mA followed by 15-s of constant current stimulation at 1.5 mA and 10 s ramp-down. Hence, sham NITESGON only lasted 15 s (as opposed to 20 min in the active group).Participants were scanned immediately before, during, and immediately after the NITESGON stimulation. The researcher who controlled the NITESGON device was not blinded to the stimulation group, but the participant was blinded to which stimulation group they were placed in. Sixteen participants received active NITESGON and 16 received sham NITESGON.A functional connectivity analysis was performed using the CONN toolbox. The ROI considered in the analysis were the right hippocampus (based on previous findings ), LC, anWe conducted an rALFF analysis for the LC, VTA, and hippocampus. The time series for each voxel of each ROI was transformed to the frequency domain and the power spectrum was then obtained. Since the power of a given frequency is proportional to the square of the amplitude of this frequency component, the square root was calculated at each frequency of the power spectrum and the averaged square root was obtained across 0.01–0.17 Hz at each voxel. This averaged square root was taken as the rALFF . The rALr-value converted to Fisher’s Z-transformed coefficients were used for further statistical analyses. The Z-transformed connectivity weights were compared between the active and sham groups for the LC and hippocampus, LC and VTA, and VTA and hippocampus, respectively, using a repeated measures ANOVA. A simple contrast analysis was applied to compare the different in active and sham condition using a Bonferroni correction.In addition, the average BOLD time series across all voxels within the ROIs were extracted from the smoothed functional images. A partial correlation analysis was performed, and the resulting Participants were 24 healthy, right-handed, native-English speaking adults  with a similar educational background. Participants were screened and enrolled similar to Experiment 1.Active NITESGON stimulation consisted of a ramp-up period of 5 s, followed by constant current of 1.5 mA for 20 min and ramp-down period of 5 s. Sham NITESGON only consisted of a ramp-up period of 5 s to reach the intensity of 1.5 mA and an immediate ramp-down period of 5 s. Twelve participants received active NITESGON and 12 participants received sham NITESGON.2 Quick Cap configured per the International 10–20 placement system with the midline reference located at the vertex and the ground electrode located at AFZ using the Neuroscan Scan 4.5 software. The impedance on each electrode was maintained at less than 5 kΩ. The data were sampled using the Neuroscan Synamps2 amplifier at 500 Hz with online band-pass filtering at 0.1–100 Hz. Data were preprocessed using Matlab and EEGLAB in a manner similar to the original paper that showed a relationship between ERP and LC–noradrenergic arousal function  was recorded in real time using a binocular Basler Dart Near Infrared (NIR) cameras. The lenses have a fixed focal length of 8 mm with an M12x0.5 body. The images were recorded at a frame rate of 120 Hz. There is a constant NIR illumination of the eye (850 nm) and the cameras are equipped with a ‘daylight cut filter’ which passes NIR and blocks any wavelengths below ~800 nm. Surface-mounted LEDs were used for light stimulation and the cameras were all mounted on a single eyepiece, which communicated with a Windows laptop through a USB 3.0 cable. Each color was shown for 200 ms first in the left eye and then in the right eye with an ISI of 8 s. This left–right trial was repeated three times for each color. The average total duration of the procedure was 2 min per participant. The participant was requested to focus on a point inside the eyepiece and open their eyes as wide as possible. They were asked to avoid rapid and frequent blinking of eyes, movement of eyes, and specifically to avoid blinking during stimulus presentation. The videos were then post-processed to obtain the dilation of the pupil. The pupil was extracted as an ellipse from each frame with a segmentation algorithm and the diameter was calculated from the average of the major and minor axes of the resulting pupil ellipse. The difference between the maximum dilation and maximum constriction of both pupils in response to each color for every trial was calculated for each person immediately before and after NITESGON. The pupil size was measured as a direct response to the NITESGON and did not include a specific task.Participants performed the auditory oddball task twice, once immediately before and once immediately after the NITESGON session. In addition, saliva and pupil dilation were also collected immediately before and immediately after the NITESGON session. The reason why we collected auditory oddball task and pupil size before and after NITESGON is to avoid a direct effect of the learning taks or inference of the current sent to the scalp. Participants were randomly assigned to the active or sham NITESGON group. The researcher who controlled the NITESGON device was not involved in instructing the participant; this was performed by a second researcher who was blind to the stimulation protocol.EEG data were compared using a repeated measures ANOVA with groups (active vs. sham) and condition (deviant vs. standard) as the between-subjects variable, and the peak-to-peak amplitude before and after stimulation as the within-subjects variable. A simple contrast analysis was applied to compare specific effects using a Bonferroni correction.We conducted a repeated measures ANOVA with group (active vs. sham) as between-subjects variable, and the average eye blink rate before and after stimulation as within-subjects variable. A simple contrast analysis was applied to compare specific contrasts using a Bonferroni correction.Using the pupil size, we conducted a one-way ANOVA with groups as group variable and difference in pupil size before and after NITESGON as the dependent variable.Using the saliva collected via the passive drool method, sAA levels were measured which were compared between the groups as detailed in Experiment 1.Pearson correlations were calculated between the difference in sAA, peak-to-peak P3b amplitude and sEBR, pupil size before and after NITESGON stimulation.Participants were 20 right-handed adults , half of who were selected due to their medical record indicating they were taking flupentixol (0.5 mg)/melitracen (10 mg) (Deanxit), a DA antagonist  , for theAssociative memory performance was measured using the same computerized Swahili–English verbal paired-associative learning task used in Experiment 1, however, the English words were replaced by Dutch words.All participants received active NITESGON immediately following the word-association task on visit 1 using the following parameters: a 5-s ramp-up period, followed by a constant current of 1.5 mA for 25 min, and finished with a 5-s ramp-down period.As experiment suggests DA plays a vital role in memory consolidation, Experiment 8 is a follow-up experiment to cross validate these findings. We opted for the parallel design not including a control condition, as the aim of this study is to see if DA antagonist mediates the memory effect induced by NITESGON, rather than the effect of NITESGON or the interaction between sleep and NITESGON on memory.Eligible participants were scheduled for two visits to complete the study. Visit 1 consisted of the word-association task followed by active NITESGON stimulation. Participants were asked to refrain from studying or searching for the learned word pairs throughout the week. Participants returned 3–4 days after their first visit for memory testing to measure possible (long-term) effects on associative memory performance, but did not receive NITESGON, nor were they able to review word pairs.For visit 1 learning, a one-way ANOVA was conducted with the cumulative learning rate over the different study periods as the dependent variable and two groups (antagonist or no antagonist) as between-subjects variable. To look at the memory effect  7 days after learning, we applied a one-way ANOVA with group as the between-subjects variable and correctly recalled words as dependent variable.χ2 analysis was used to determine if there was a difference between what stimulation participants received compared to what participants expected.To determine if the stimulation for all experiments was well blinded, all participants who participated in Experiments 1–7 were asked to complete a single-response questionnaire after the conclusion of the NITESGON procedure. Here, participants were asked to guess if they thought they were placed in the active or control group. A  This is a landmark study showing that non-invasive transcutaneous electrical stimulation of the greater occipital nerve (NITESGON) can improve long-term memory. The authors provide compelling evidence that applying NITESGON during learning causally influences memory behavior. This method is novel and the work presented herein will be valuable to a broad range of scientists and clinicians interested in manipulations to improve memory and cognition more broadly. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Floris de Lange as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Krista L Wahlstrom (Reviewer #2).Thank you for submitting your article "Making memories last: The peripheral effect of direct current stimulation on strengthening memories" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Thank you for an experimental tour-de-force! This manuscript has several strengths and the data definitely point to the result that this stimulation method and protocols do improve memory. That said, the mechanisms are oversold in the manuscript. Both reviewers agreed that several of the claims made in the paper were not warranted by the data. While the inclusion of several separate experiments is impressive and thorough, it also does limit the conclusions that can be made across experiments. A thorough and successful revision will roll back interpretational claims that are not justified by the approach or results. The critical ones to attend to are outlined clearly and thoughtfully in both reviews so I will not restate them in detail here. It is absolutely critical that the authors be upfront about the lack of specificity in their proxy measures of DA and NA activity (eyeblink and sAA). They are both indirect measures and deserve less emphasis than is currently there. In addition to addressing the reviewer comments in full, I want to highlight that the revision should address the lack of control/sham groups in Exp 5 and 8 to justify and explain how the lack of a control group changes their interpretation of the findings. The revision should better differentiate Exp 3 and 4, how do they differ? It was also noted that the interpretation of an interference effect needed to be further justified by making it clear what behavioral measure of interference was used.Reviewer #1 (Recommendations for the authors):Missing background information:It would strengthen the paper to describe the central and autonomic pathways engaged by NITESGON. Elaborating on this neuroanatomy is important because this stimulation method doesn't target the LC specifically or directly but rather upstream targets like the vagus nerve and NTS. While the data do align with the a priori prediction that it engages learning-relevant processes in the LC, the paper would be more accessible if there was a deeper discussion of NITESGON and how it affects brainstem pathways.Considerations for correlations:The individual differences correlations are compelling. One key strength of the paper is how consistently some of these findings replicate. But it is also important to include separate correlations for the sham and stimulation groups. A qualitative comparison suggests that, in some cases, stimulation might decorrelate the two variables of interest . This makes it difficult to interpret the linearity of the effect. Qualitatively, the between-group differences appear to obscure within-group effects, which casts some doubt on whether the relationship is linear and, importantly, remains linear in the stimulation group alone. The sample sizes are also quite small, which makes the regressions sensitive to outliers. Please report whether the correlations meet assumptions of linearity and if they are violated, adopt methods that are less sensitive to outliers .To strengthen the argument that stimulation-induced increases in sEBR and sAA relate to oddball-evoked responses , it would also be useful to account for within-subject effects. Please use subtraction scores to account for these differences .Spatial resolution of MRI:The LC is notoriously difficult to image due to its small size (~1-2mm across in humans) and susceptibility to MR signal artifacts from cardiac pulsation and partial volume effects from the fourth ventricle . The current MRI parameters are sub-optimal for imaging the LC due to their low spatial resolution of 3 x 3 x 4 mm3 as well as the very large smoothing kernel (8mm). To help make claims about the LC, it is critical to re-analyze this data using no smoothing to better approximate the size of the LC. In its current form, the data may reflect the contribution of signals from other neighboring nuclei and brainstem regions engaged by NITESGON. In addition to performing this analysis, these issues should still be raised as important caveats in the discussion.In addition, global signal regression in resting-state fMRI has courted much controversy and may lead to spurious correlations. It would be useful to demonstrate that the results still hold without using this preprocessing step.It's been shown that VNS can enhance widespread effects on the BOLD signal across the brain . The authors observe significant increases in activation across all regions of interest in this study. To verify the specificity of stimulation effects, it would be helpful to also report the effects in at least one control region, such as the primary motor cortex. Again, VNS is a different technique than NITESGON, so more broadly it would help to differentiate whether the effects of these methods on neural function would differ. This would ground the current findings in similar prior work and highlight the advantages of using this specific method of stimulation to boost memory.Considerations about physiological measures and interpretations:On Page 15, it is hypothesized that sEBR would correlate with pupil diameter. However, there is no mention of pupillometry data or analysis throughout the paper. This is unfortunate because pupil diameter is perhaps the best indirect proxy of LC activity . Is this data available? That would provide more convincing evidence of a link between learning and noradrenergic signaling.On a related note, it has been previously shown that tVNS, which also engages the vagus nerve, elicits increases in sAA but does not affect pupil diameter . Furthermore, sAA is not always closely coupled with other indices of norepinephrine release . Together, these findings raise the possibility that sAA isn't a direct marker of (only) LC activation. Unfortunately, a direct relationship can't be verified in this study because LC activity and sAA weren't collected in the same study/sample. Please address the caveat that sAA might not relate directly to LC activation in the discussion.While spontaneous blink rate has been increasingly linked to dopamine, these studies typically examine the relationship between sEBR and striatal dopamine . By comparison, little work has explored the link between sEBR and hippocampal memory processes, and there is no way to explicitly link them in the current dataset. Prior work on sEBR also typically link sEBR phenomena to prefrontal executive functions , such as working memory or updating . Thus, sEBR in effects in Experiment 7 may relate to attentional effects, as the oddball paradigm suggests, but not necessarily memory encoding. There is also some evidence that the sEBR and DA levels may be unrelated . While I find the current findings interesting, it's a very big stretch to interpret blink rate as reflecting LC dopamine. Like sAA, blink rate could reflect other neural processes that are engaged by NITESGON but aren't driven by a common underlying mechanism. This framing/interpretation needs to be tempered significantly. It would be more appropriate to frame the goals of that section as examining possible relationships with DA, but not LC-DA specifically. It's fair to speculate they may be related, but that is very much open to interpretation and belongs in the discussion.Figures:1) Correctly recall should be "correctly recalled".2) The use of blue and red colors for both sham and stimulation conditions as well as before and after is very confusing. Please use different colors and keep consistent throughout.3) In Figure 6i-m, keep the axes consistent  for clarity.4) Similarly, for Figures 3 and 4, keep A and B consistent (lines or bars) since they convey similar things.5) The “δ deviant” etc. y-axes aren’t very descriptive. Please use clear variable names pertaining to that measure.Reviewer #2 (Recommendations for the authors):1) In line 68 “LC-NA pathway” is abbreviated before those abbreviations are written out as text.2) Citation needed in line 95 when the authors state "as shown before…".3) Citation needed in line 340 – "However, recent research revealed that mainly LC DA…".4) In line 388 it would be helpful for the authors to clarify what the memory test 3 to 4 days after initial learning is and if there are results for those tests. Similarly, in Experiment 8, a justification for the 3-4 day memory test instead of the 7-day test would be helpful.5) Typo in lines 440 and 464 – "NTESGON".6) Line 753 – Would read easier from the perspective of the active stimulation group. i.e. "Experiment 3 had all participants receive NITESGON during each study period of task 1…".7) Line 754 – shouldn't the active NITESGON stimulation be during the first task for this experiment?8) It would be helpful to include individual participant data points in the bar graphs across all experiments/figures.9) In Figure 1D there is a green sham data point in the panel legend.eLife. Your revised article has been evaluated by Floris de Lange (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Making memories last using the peripheral effect of direct current stimulation." for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:The reviewers both commend the authors on a very responsive revision which steps back from making causal claims that are not justified and the manuscript is more transparent now about shortcomings of the design. That said, there are some outstanding concerns that should be addressed.The first concerns the statistical reporting of correlations that Reviewer 1 had recommended initially and still requires attention. It is important for the authors to report their correlations separately by group or to include a group as a covariate in the statistical analyses. If the correlations uncouple within a group, this should be reported and discussed.Second, there was confusion about pupil data acquisition and when and how they were acquired. I will point your attention to Reviewer 1's specific comment here.Third, it is important that your discussion be as specific as possible about the manipulation and the effects noted. For example Reviewer 2 points out, and correctly so, that the dependent measure of # of words recalled after stimulation may or may not reflect interference effects, as is claimed. It is recommended that the authors use a specific measure of interference if allowable, or refrain from claiming their measure(s) reflect interference.See the reviewers' specific comments below:Reviewer #1 (Recommendations for the authors):First, I commend the authors on the substantial work they invested in this revision. They have addressed most of my comments and this was no small effort. I especially appreciate that they have acknowledged the lack of causality and direct associations between neuromodulators and some of these indirect biomarkers. However, I do have a few outstanding concerns that I believe warrant consideration prior to publication.1) The pupil analyses were a little puzzling. It would more sensible to measure discrete pupil dilations to the deviant vs. standard stimuli, as LC responses are most often measured to reflect "cognitive activity" rather than luminance. The LC will be stimulated by blue light, but it would be stronger to report oddball-evoked effects. I'm not sure if I misunderstood whether the pupil wasn't recorded during the task itself.2) I'd still recommend reporting separate correlations by group. Many of the correlation effects appear to uncouple in the stimulation condition, which could alter some interpretations of the data. If this happens, additional discussion is warranted.3) The addition of the "before" and "after" (blue/yellow colors) in some of the keys was a useful change. However, these names could be more descriptive. It's not readily apparent what these mean when the other variable also describes timepoints .4) There are quite a number of grammatical and spelling errors throughout, which I won't point out individually. The paper should be proofread again and revised before final acceptance.5) It would be useful to foreground the "competition" effects of tag-and-capture in the Results section for the interference studies. This is described well in the discussion, but the same point should be repeated higher up. Otherwise, this buries the lead for the reader. Essential revisions:Thank you for an experimental tour-de-force! This manuscript has several strengths and the data definitely point to the result that this stimulation method and protocols do improve memory. That said, the mechanisms are oversold in the manuscript. Both reviewers agreed that several of the claims made in the paper were not warranted by the data. While the inclusion of several separate experiments is impressive and thorough, it also does limit the conclusions that can be made across experiments. A thorough and successful revision will roll back interpretational claims that are not justified by the approach or results. The critical ones to attend to are outlined clearly and thoughtfully in both reviews so I will not restate them in detail here.Thank you for an experimental tour-de-force! This manuscript has several strengths, and the data definitely point to the result that this stimulation method and protocols do improve memory. That said, the mechanisms are oversold in the manuscript. Both reviewers agreed that several of the claims made in the paper were not warranted by the data. While the inclusion of several separate experiments is impressive and thorough, it also does limit the conclusions that can be made across experiments. A thorough and successful revision will roll back interpretational claims that are not justified by the approach or results. The critical ones to attend are outlined clearly and thoughtfully in both reviews so I will not restate them in detail here.It is absolutely critical that the authors be upfront about the lack of specificity in their proxy measures of DA and NA activity (eyeblink and sAA). They are both indirect measures and deserve less emphasis than is currently there.Upon consideration of your comment, we have included a paragraph in the Discussion section highlighting limitations that exist within the research. Within this paragraph, we have emphasized that the indirect proxy measures of sAA and sEBR are associated with being substantially variable and that sAA may also reflect changes in monoamine neurotransmitters of other brain regions. Here we advise that readers take caution when interpreting the results due to these limitations and lack of specificity in the proxy measures.Please see the newly added text from the Discussion section on page 22 below:“Although the use of indirect proxy measures, such as sAA for NA activity and sEBR for DA activity, enabled the tracking of LC-NA activity changes from baseline measurements and demonstrated the potential of an LC-DA relationship, caution must be advised when interpreting results considering these proxy measures are affiliated with limitations, such as being substantially variable, as well as the potential of other brain regions and monoamine neurotransmitters being associated with changes seen in sAA concentration levels(82), an enzyme that is provoked by both central parasympathetic and sympathetic nervous system activation, including acute stress responses(83). Additionally, although sEBR has been increasingly linked to DA, it has been defined as a more viable measure of striatal DA activity . At the same time, our research suggests that sEBR and DA are related as well as with the peak-to-peak amplitude for deviant suggesting at least a possible common underlying mechanism. Lastly, it must be recognized that the effects of NITESGON obtained could be explained by activating other neuromodulatory mechanisms. Previous animal research indicates that peripheral nerve stimulation, such as vagus nerve stimulation, also activates the dopaminergic , serotonergic (89), and cholinergic (90) pathways, all of which play an essential role in inducing long-term plasticity changes related to memory consolidation(91). Further studies regarding the role of additional neuromodulators would be worthwhile.”In addition to addressing the reviewer comments in full, I want to highlight that the revision should address the lack of control/sham groups in Exp 5 and 8 to justify and explain how the lack of a control group changes their interpretation of the findings.Upon review of your comment, we have addressed the lack of control/sham groups in Experiment 5 and 8 in the Discussion section when acknowledging the limitations of the research. Additionally, we have pointed out that including control/sham groups in these experiments would influence our interpretation of the results by increasing our confidence of NITESGON’s memory-enhancing effect not being sleep dependent but rather being dependent upon DA receptor activity.Please see the newly added text from the Discussion section on pages 21-22 below:“Furthermore, the recall-only memory testing occurred at the 12-hour point rather than 7-days later, allowing us to conclude that the observed effect seen 12-hours later was not affected by sleep; however, it remains unclear whether the 7-day long-term memory effects of NITESGON are sleep-dependent. Moreover, it must also be acknowledged that Experiments 5 and 8 did not include a control-sham stimulation group, thus limiting the interpretation of these two experimental findings. Control-sham stimulation groups would increase our confidence in our findings that NITESGON’s memory-enhancing effects depend not on sleep but on DA receptor activity.”The revision should better differentiate Exp 3 and 4, how do they differ?In order to clarify what the differences were between Experiment 3 and Experiment 4, we added text in the paragraph introducing Experiment 4 clarifying that the object-location task used in Experiment 3 was replaced in Experiment 4 with a Japanese-English verbal associative learning task.Please see the paragraph from the Experiment 4 subsection on page 10 below:“Experiments 2 and 3 revealed both retroactive and proactive memory effects 7-days after initial learning of the two tasks. To further explore if NITESGON is linked to behavioral tagging and evaluate if interference impacts NITESGON as the strong stimulus, Experiment 4 removed the object-location task used in Experiments 2 and 3 and replaced it with a Japanese-English verbal associative learning task similar to the Swahili-English verbal associative task. Considering how memory formation and persistence are susceptible to interference occurring pre-and post-encoding(37-39) and are heavily influenced by commonality amongst the learned and intervening stimuli(40); it is believed that conducting two consecutive, like-minded word-association  tasks will result in one’s consolidation process interfering with that of the other(41). Considering how our previous experiments suggest the effect obtained by NITESGON improves the consolidation of information via behavioral tagging, it is possible that NITESGON on the first task might help reduce the overall interference effect on the second task.”It was also noted that the interpretation of an interference effect needed to be further justified by making it clear what behavioral measure of interference was used.Upon review of this comment, we thought that it was best to modify the paper in two ways. First, we explained in further detail that comparing the percentage of correctly recalled word pairs on the second task 7-days after learning from the percentage of correctly recalled word pairs on the first task 7-days after learning was done to measure for an interference effect.Please see the adapted text from the Experiment 4 subsection on page 11 below:“Upon assessment for a potential interference effect, the active group displayed no significant difference in how many words participants were able to recall between the first and the second task (difference:.76 ±4.93) , whereas the sham group demonstrated the first task rendered an interference effect on the second task (difference: 5.16 ±5.99) .”Additionally, the methods section describing how the interference effect was calculated was changed. The newly edited text better explains that the percentage of words pairs learned were subtracted from one another to measure the significance of interference one may have potentially had on the other.Please see the amended text in the Methods section on page 38 below:“In addition, an interference effect was calculated by subtracting the percentage of correctly recalled word pairs on the second task 7-days after learning from the percentage of correctly recalled word pairs on the first task 7-days after learning. This number gave a proxy of interference.”Reviewer #1 (Recommendations for the authors):Missing background information:It would strengthen the paper to describe the central and autonomic pathways engaged by NITESGON. Elaborating on this neuroanatomy is important because this stimulation method doesn't target the LC specifically or directly but rather upstream targets like the vagus nerve and NTS. While the data do align with the a priori prediction that it engages learning-relevant processes in the LC, the paper would be more accessible if there was a deeper discussion of NITESGON and how it affects brainstem pathways.Per your suggestion, we have provided a description of the potential pathway stimulation may take to activate the LC-NA system.Please see the newly added text from the Introduction section on page 3 below:“In addition, our recent work suggests that non-invasive transcutaneous electrical stimulation of the greater occipital nerve (NITESGON) using direct current utilizes a pathway that arises from the C2 spinal nerve to establish communication gateways from the periphery to the brain via afferent fibers that project to the brainstem and synapse onto the nucleus tractus solitarius (NTS); various rodent studies have used anterograde neuronal tracers to demonstrate these connections (8-11). From the NTS, the information is then integrated amongst networks within the complex reticular formation and relayed across the brainstem through major cortical and subcortical regions(12).”We have also acknowledged how the effects of NITESGON may be explained via the activation of other neuromodulatory pathways via peripheral nerve stimulation. To elaborate, we draw parallels with how vagus nerve stimulation can activate the dopaminergic, serotonergic, and cholinergic pathways, all of which play a role in plasticity changes.Please see the newly added text from the Discussion section on page 22 below:“Lastly, it must be recognized that the effects of NITESGON obtained could be explained by activating other neuromodulatory mechanisms. Previous animal research indicates that peripheral nerve stimulation, such as vagus nerve stimulation, also activates the dopaminergic(85), serotonergic(86), and cholinergic(87) pathways, all of which play an essential role in inducing long-term plasticity changes related to memory consolidation(88). Further studies regarding the role of additional neuromodulators would be worthwhile.”Considerations for correlations:The individual differences correlations are compelling. One key strength of the paper is how consistently some of these findings replicate. But it is also important to include separate correlations for the sham and stimulation groups. A qualitative comparison suggests that, in some cases, stimulation might decorrelate the two variables of interest . This makes it difficult to interpret the linearity of the effect. Qualitatively, the between-group differences appear to obscure within-group effects, which casts some doubt on whether the relationship is linear and, importantly, remains linear in the stimulation group alone. The sample sizes are also quite small, which makes the regressions sensitive to outliers. Please report whether the correlations meet assumptions of linearity and if they are violated, adopt methods that are less sensitive to outliers .We understand the concern and included for correlation the Spearman rank in addition to the Pearson to get a better idea of all correlations. We implemented a supplement figure just the ranks to present the Spearman correlation. See supl Figure 1 and text.To strengthen the argument that stimulation-induced increases in sEBR and sAA relate to oddball-evoked responses , it would also be useful to account for within-subject effects. Please use subtraction scores to account for these differences .We included the subtraction score for both sEBR and sAA in the paper.“The subtraction scores for the sEBR  and sAA , revealed a significant difference between the active and the sham group further indicatin a stimulation-induced increases in sEBR and sAA.”Spatial resolution of MRI:The LC is notoriously difficult to image due to its small size (~1-2mm across in humans) and susceptibility to MR signal artifacts from cardiac pulsation and partial volume effects from the fourth ventricle . The current MRI parameters are sub-optimal for imaging the LC due to their low spatial resolution of 3 x 3 x 4 mm3 as well as the very large smoothing kernel (8mm). To help make claims about the LC, it is critical to re-analyze this data using no smoothing to better approximate the size of the LC. In its current form, the data may reflect the contribution of signals from other neighboring nuclei and brainstem regions engaged by NITESGON. In addition to performing this analysis, these issues should still be raised as important caveats in the discussion.In addition, global signal regression in resting-state fMRI has courted much controversy and may lead to spurious correlations. It would be useful to demonstrate that the results still hold without using this preprocessing step.We understand the concern and included an additional analysis not using a smoothing kernel. Our result remains more or less the same. We included this additional analysis in supplement material and at this to results and Discussion section.“To further confirm our data, we replicated our analysis by not including a smoothing kernel, showing similar results for the LC, VTA and hippocampus . Furthermore, we included two control areas, the left inferior parietal cortex, where we do not expect to see any changes .”“One note, is that we need to be careful with the results of the LC, as this area is notoriously difficult to image due to its small size (~1-2mm across in humans) and susceptibility to MR signal artifacts from cardiac pulsation and partial volume effects from the fourth ventricle (78). Therefore, we re-analyzed our data using no smoothing to better approximate the size of the LC, showing similar results.”It's been shown that VNS can enhance widespread effects on the BOLD signal across the brain . The authors observe significant increases in activation across all regions of interest in this study. To verify the specificity of stimulation effects, it would be helpful to also report the effects in at least one control region, such as the primary motor cortex. Again, VNS is a different technique than NITESGON, so more broadly it would help to differentiate whether the effects of these methods on neural function would differ. This would ground the current findings in similar prior work and highlight the advantages of using this specific method of stimulation to boost memory.We include a different area not showing effect.Considerations about physiological measures and interpretations:On Page 15, it is hypothesized that sEBR would correlate with pupil diameter. However, there is no mention of pupillometry data or analysis throughout the paper. This is unfortunate because pupil diameter is perhaps the best indirect proxy of LC activity . Is this data available? That would provide more convincing evidence of a link between learning and noradrenergic signaling.We included these findings in both results and method section.“Experiment 7. Potential relationship between NITESGON-LC and dopamine.The previous experiment revealed activity changes in both the LC and hippocampus as well as increased connectivity between the LC and hippocampus both during and after NITESGON. Conversely, the VTA did not show changes in activity after stimulation or connectivity changes between the VTA and hippocampus during or after stimulation. However, activity changes in the VTA during NITEGSON were detected. Previous animal research has identified selective neuronal connections between the LC and VTA, implying an interaction between the LC and VTA during NITESGON may exist(31).[…]As previous research already showed that pupil size is an important proxy for LC mediate activity, we also look at pupil size. Our data show that a significant difference in pupil size after stimulation with baseline correction for active stimulation (2.28 +1.90) in comparison to sham (-3.59 +4.80) stimulation . Furthermore, a significant positive correlation was between pupil size and sEBR  and between pupil size and sAA .”On a related note, it has been previously shown that tVNS, which also engages the vagus nerve, elicits increases in sAA but does not affect pupil diameter . Furthermore, sAA is not always closely coupled with other indices of norepinephrine release . Together, these findings raise the possibility that sAA isn't a direct marker of (only) LC activation. Unfortunately, a direct relationship can't be verified in this study because LC activity and sAA weren't collected in the same study/sample. Please address the caveat that sAA might not relate directly to LC activation in the discussion.Upon consideration of your comment, we have included a paragraph in the Discussion section highlighting limitations that exist within the research. Within this paragraph, we have emphasized that the indirect proxy measures of sAA is associated with being substantially variable and that sAA may also reflect changes in monoamine neurotransmitters of other brain regions. Here we advise that readers take caution when interpreting the results due to these limitations and lack of specificity in using sAA as a proxy measure.Please see the newly added text from the Discussion section on page 22 below:“Although the use of indirect proxy measures, such as sAA for NA activity and sEBR for DA activity, enabled the tracking of LC-NA activity changes from baseline measurements and demonstrated the potential of an LC-DA relationship, caution must be advised when interpreting results considering these proxy measures are affiliated with limitations, such as being substantially variable, as well as the potential of other brain regions and monoamine neurotransmitters being associated with changes seen in sAA concentration levels(80), an enzyme that is provoked by both central parasympathetic and sympathetic nervous system activation, including acute stress responses(81).”While spontaneous blink rate has been increasingly linked to dopamine, these studies typically examine the relationship between sEBR and striatal dopamine . By comparison, little work has explored the link between sEBR and hippocampal memory processes, and there is no way to explicitly link them in the current dataset. Prior work on sEBR also typically link sEBR phenomena to prefrontal executive functions , such as working memory or updating . Thus, sEBR in effects in Experiment 7 may relate to attentional effects, as the oddball paradigm suggests, but not necessarily memory encoding. There is also some evidence that the sEBR and DA levels may be unrelated . While I find the current findings interesting, it's a very big stretch to interpret blink rate as reflecting LC dopamine. Like sAA, blink rate could reflect other neural processes that are engaged by NITESGON but aren't driven by a common underlying mechanism. This framing/interpretation needs to be tempered significantly. It would be more appropriate to frame the goals of that section as examining possible relationships with DA, but not LC-DA specifically. It's fair to speculate they may be related, but that is very much open to interpretation and belongs in the discussion.Upon consideration of your comment, we have included a paragraph in the Discussion section highlighting limitations that exist within the research. Within this paragraph, we have emphasized that the indirect proxy measures of sEBR is associated with being substantially variable. Here we advise that readers take caution when interpreting the results due to these limitations and lack of specificity in the proxy measures.Please see the newly added text from the Discussion section on page 22 below:“Additionally, although sEBR has been increasingly linked to DA, it has been defined as a more viable measure of striatal DA activity. At the same time, some evidence suggests that sEBR and DA levels may be unrelated, thus requiring further validation as a behavioral proxy measure.”Figures:1) Correctly recall should be "correctly recalled".We changed this in all figures.2) The use of blue and red colors for both sham and stimulation conditions as well as before and after is very confusing. Please use different colors and keep consistent throughout.We change this.3) In Figure 6i-m, keep the axes consistent  for clarity.We included these changes.4) Similarly, for Figures 3 and 4, keep A and B consistent (lines or bars) since they convey similar things.We made sure that it is consistent.5) The "δ deviant" etc. y-axes aren't very descriptive. Please use clear variable names pertaining to that measure.We explain this in the figure legends.Reviewer #2 (Recommendations for the authors):1) In line 68 "LC-NA pathway" is abbreviated before those abbreviations are written out as text.Thank you for spotting our mistake in abbreviating “LC-NA pathway” before we wrote out the abbreviations. We have edited the text so that “LC-NA pathway” is now written out and the next appearance of the term is abbreviated.Please see the corrected text on page 4 below:“The neural mechanism that controls this novelty response is the locus coeruleus-noradrenaline (LC-NA) pathway. Animal research further indicates that direct electrical stimulation of the locus coeruleus (LC) modulates hippocampal synaptic consolidation(26-28).”2) Citation needed in line 95 when the authors state "as shown before…".Thank you for noticing a citation was missed and bringing it to our attention. We have edited the text so that the proper citation is included.Please see the citation added on page 5 below:“Seeing that NITESGON activates the LC pathway, which plays an important role in memory consolidation, we hypothesize that participants will be able to establish long-term memories upon modulating the LC both during learning, as shown before, as well as immediately after learning.”3) Citation needed in line 340 – "However, recent research revealed that mainly LC DA…".Thank you for noticing a citation was missed and bringing it to our attention. We have edited the text so that the proper citation is included.Please see the citation added on page 15 below:“However, recent research revealed that mainly LC DA mediates post-encoding memory enhancement in the hippocampus, while the VTA does not respond to arousal .”4) In line 388 it would be helpful for the authors to clarify what the memory test 3 to 4 days after initial learning is and if there are results for those tests. Similarly, in Experiment 8, a justification for the 3-4 day memory test instead of the 7-day test would be helpful.Upon review of the paper and your comment, we have added clarification to the sentence by indicating that the memory test 3 to 4 days after initial learning was the recall-only memory test. The justification for including the memory test 3-4 days after initial learning rather than 7 days after initial learning lies within participants of Experiment 8 falling in an older age population. The 3-to-4-day separation between initial learning and memory testing allowed for a fixed scheduling practice and enabled a more convenient scheduling policy that was easier for the population to subscribe to.Please see the amended text from page 17 below:“To test this hypothesis, and confirm previous findings, Experiment 8 conducted the recall-only memory test 3 to 4-days after initial learning of the word-association task.”5) Typo in lines 440 and 464 – "NTESGON".Thank you for catching the spelling mistakes on line 440 and line 464 and bringing them to our attention. We have reviewed the spelling errors and have corrected them to “NITESGON.”Please see the corrected text on pages 19 and 22 below:“In addition, we demonstrated changes in sAA immediately after NITESGON that correlate with memory recall 7-days later.”“In conclusion, our work provides evidence that NITESGON is involved in the consolidation of information rather than encoding.”6) Line 753 – Would read easier from the perspective of the active stimulation group. i.e. "Experiment 3 had all participants receive NITESGON during each study period of task 1…".Per your suggestion, we have edited the wording so that it is described from the perspective of the active stimulation group and is easier for the reader to understand.Please see the new wording on page 34 below:“Differing from Experiment 2, Experiment 3 had all participants receive active NITESGON during each study period of task 1 as opposed to task 2.”7) Line 754 – shouldn't the active NITESGON stimulation be during the first task for this experiment?Thank you for spotting and pointing out the error on line 754. We have edited the paper so that line 754 has been corrected and states that NITESGON stimulation was administered during the first task.Please see the corrected text on page 34 below:“12 participants received active NITESGON and 12 participants received sham NITESGON during the first task.”8) It would be helpful to include individual participant data points in the bar graphs across all experiments/figures.We include this in the figures.9) In Figure 1D there is a green sham data point in the panel legend.We resolve this issue.The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:The reviewers both commend the authors on a very responsive revision which steps back from making causal claims that are not justified and the manuscript is more transparent now about shortcomings of the design. That said, there are some outstanding concerns that should be addressed.The first concerns the statistical reporting of correlations that Reviewer 1 had recommended initially and still requires attention. It is important for the authors to report their correlations separately by group or to include a group as a covariate in the statistical analyses. If the correlations uncouple within a group, this should be reported and discussed.We added the correlations for each group separately for each of the experiments. A description was included for each experiment in the Results section. All figures include also the correlation and regression line for each group separately.Second, there was confusion about pupil data acquisition and when and how they were acquired. I will point your attention to Reviewer 1's specific comment here.We clarify this in the paper. “Pupil dilation. The response of the pupil to three types of light stimulation  was recorded in real-time using a binocular Basler Dart Near Infrared (NIR) cameras. The lenses have a fixed focal length of 8 mm with an M12x0.5 body. The images were recorded at a frame rate of 120 Hz. There is a constant NIR illumination of the eye (850 nm) and the cameras are equipped with a “daylight cut filter” which passes NIR and blocks any wavelengths below ~800 nm. Surface-mounted LEDs were used for light stimulation and the cameras were all mounted on a single eyepiece, which communicated with a Windows laptop through a USB 3.0 cable. Each color was shown for 200 ms first in the left eye and then in the right eye with an ISI of 8 seconds. This left-right trial was repeated three times for each color. The average total duration of the procedure was two minutes per participant. The participant was requested to focus on a point inside the eyepiece and open their eyes as wide as possible. They were asked to avoid rapid and frequent blinking of eyes, movement of eyes, and specifically to avoid blinking during stimulus presentation. The videos were then post-processed to obtain the dilation of the pupil. The pupil was extracted as an ellipse from each frame with a segmentation algorithm and the diameter was calculated from the average of the major and minor axes of the resulting pupil ellipse. The difference between the maximum dilation and maximum constriction of both pupils in response to each color for every trial was calculated for each person immediately before and after NITESGON. The pupil size was measured as a direct response to the NITESGON and did not include a specific task.”Third, it is important that your discussion be as specific as possible about the manipulation and the effects noted. For example Reviewer 2 points out, and correctly so, that the dependent measure of # of words recalled after stimulation may or may not reflect interference effects, as is claimed. It is recommended that the authors use a specific measure of interference if allowable, or refrain from claiming their measure(s) reflect interference.We understand the concern and apologies for the confusion. We recalculated the interference effect as suggested by reviewer 2.See the reviewers' specific comments below:Reviewer #1 (Recommendations for the authors):First, I commend the authors on the substantial work they invested in this revision. They have addressed most of my comments and this was no small effort. I especially appreciate that they have acknowledged the lack of causality and direct associations between neuromodulators and some of these indirect biomarkers. However, I do have a few outstanding concerns that I believe warrant consideration prior to publication.1) The pupil analyses were a little puzzling. It would more sensible to measure discrete pupil dilations to the deviant vs. standard stimuli, as LC responses are most often measured to reflect "cognitive activity" rather than luminance. The LC will be stimulated by blue light, but it would be stronger to report oddball-evoked effects. I'm not sure if I misunderstood whether the pupil wasn't recorded during the task itself.We clarify this in the paper. “Pupil dilation. The response of the pupil to three types of light stimulation  was recorded in real-time using a binocular Basler Dart Near Infrared (NIR) cameras. The lenses have a fixed focal length of 8 mm with an M12x0.5 body. The images were recorded at a frame rate of 120 Hz. There is a constant NIR illumination of the eye (850 nm) and the cameras are equipped with a “daylight cut filter” which passes NIR and blocks any wavelengths below ~800 nm. Surface-mounted LEDs were used for light stimulation and the cameras were all mounted on a single eyepiece, which communicated with a Windows laptop through a USB 3.0 cable. Each color was shown for 200 ms first in the left eye and then in the right eye with an ISI of 8 seconds. This left-right trial was repeated three times for each color. The average total duration of the procedure was two minutes per participant. The participant was requested to focus on a point inside the eyepiece and open their eyes as wide as possible. They were asked to avoid rapid and frequent blinking of eyes, movement of eyes, and specifically to avoid blinking during stimulus presentation. The videos were then post-processed to obtain the dilation of the pupil. The pupil was extracted as an ellipse from each frame with a segmentation algorithm and the diameter was calculated from the average of the major and minor axes of the resulting pupil ellipse. The difference between the maximum dilation and maximum constriction of both pupils in response to each color for every trial was calculated for each person immediately before and after NITESGON. The pupil size was measured as a direct response to the NITESGON and did not include a specific task.”“Procedure: Participants performed the auditory oddball task twice, once immediately before and once immediately after the NITESGON session. In addition, saliva and pupil dilation were also collected immediately before and immediately after the NITESGON session. The reason why we collected auditory oddball task and pupil size before and after NITESGON is to avoid a direct effect of the learning taks or inference of the current sent to the scalp. Participants were randomly assigned to the active or sham NITESGON group. The researcher who controlled the NITESGON device was not involved in instructing the participant; this was performed by a second researcher who was blind to the stimulation protocol.”2) I'd still recommend reporting separate correlations by group. Many of the correlation effects appear to uncouple in the stimulation condition, which could alter some interpretations of the data. If this happens, additional discussion is warranted.We added the correlations for each group separately for each of the experiments.3) The addition of the "before" and "after" (blue/yellow colors) in some of the keys was a useful change. However, these names could be more descriptive. It's not readily apparent what these mean when the other variable also describes timepoints .We included this in the paper by indicating that before or after NITESGON.4) There are quite a number of grammatical and spelling errors throughout, which I won't point out individually. The paper should be proofread again and revised before final acceptance.I asked a native speaker to double check for typo.5) It would be useful to foreground the "competition" effects of tag-and-capture in the Results section for the interference studies. This is described well in the discussion, but the same point should be repeated higher up. Otherwise, this buries the lead for the reader.We include this in the paper under experiment 4. “Experiments 2 and 3 revealed both retroactive and proactive memory effects 7-days after initial learning of the two tasks. To further explore if NITESGON is linked to behavioral tagging and evaluate if interference impacts NITESGON as the strong stimulus, Experiment 4 removed the object-location task used in Experiments 2 and 3 and replaced it with a Japanese-English verbal associative learning task similar to the Swahili-English verbal associative task. Considering that memory formation and persistence are susceptible to interference occurring pre-and post-encoding(39-41) and are heavily influenced by commonality amongst the learned and intervening stimuli(42); it is believed that conducting two consecutive, like-minded word-association  tasks will result in one’s consolidation process interfering with that of the other(43). Furthermore, research on the synaptic tag-and-capture hypothesis suggest that memory interference is the result of synaptic competition, a proposed “fight for proteins” that arises between tagged synapses amongst limited proteins that leads to one memory converting to long-term memory at the expense of the other(68). Considering how our previous experiments suggest the effect obtained by NITESGON improves the consolidation of information via behavioral tagging, it is possible that NITESGON on the first task might help reduce the overall interference effect on the second task.”