Ntn1) is highly enriched in the ventral midbrain where it is expressed in both GABAergic and dopaminergic neurons, but its function in these cell types in the adult system remains largely unknown. To address this, we performed viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis of Ntn1 in the ventral tegmental area (VTA) of adult mice. Ntn1 loss-of-function in either cell type resulted in a significant reduction in excitatory postsynaptic connectivity. In dopamine neurons, the reduced excitatory tone had a minimal phenotypic behavioral outcome; however, reduced glutamatergic tone on VTA GABA neurons induced behaviors associated with a hyperdopaminergic phenotype. Simultaneous loss of Ntn1 function in both cell types largely rescued the phenotype observed in the GABA-only mutagenesis. These findings demonstrate an important role for Ntn1 in maintaining excitatory connectivity in the adult midbrain and that a balance in this connectivity within two of the major cell types of the VTA is critical for the proper functioning of the mesolimbic system.The axonal guidance cue netrin-1 serves a critical role in neural circuit development by promoting growth cone motility, axonal branching, and synaptogenesis. Within the adult mouse brain, expression of the gene encoding ( Proper regulation of the midbrain dopamine system is essential for numerous brain functions and behavior . DisruptGenome-wide association studies and analysis of de novo mutations have strongly implicated genes regulating neuronal axon guidance in neurodevelopmental disorders . AlthougNtn1) is highly expressed throughout the central nervous system (CNS). Following this critical period global expression decreases  and probed for Ntn1, Th , and Slc32a1 . We found Ntn1 expression to be present throughout the VTA, largely localized to Th-positive neurons but also present in GABA neurons  and Slc32a1-expressing GABA neurons   . The rem neurons , or poss neurons . Immunoh neurons  confirmeNtn1 in specific cell types in the VTA, we designed a single guide RNA (sgRNA) targeting exon 2 in mice  mice (aged 8–10 weeks) bilaterally into the VTA with either AAV-FLEX-SaCas9-HA-sgNtn1 and AAV-FLEX-YFP (DAT-Cre Ntn1-cKO mice) or AAV-FLEX-SaCas9-sgRosa26 . Four to five weeks following injection, we performed immunohistochemistry for Ntn1 and Th. Ntn1 conditional knockout (cKO) resulted in a significant reduction in the proportion of VTA Th-positive cells co-labeled with Ntn1 in DAT-Cre Ntn1 cKO mice compared to controls (Ntn1 deletion in the substantia nigra (Ntn1 cKO mice compared to controls (control: 178.2 ± 12.68 and Ntn1 cKO 173.3 ± 10.75). Although, this result is consistent with Ntn1 inactivation not compromising cell viability, without a complete stereological analysis of every neuron within the VTA, we cannot definitively conclude that some cell loss did not occur.To selectively mutate  sgNtn1;  and clon sgNtn1; . To detecontrols . In contia nigra , the aveNtn1 loss of function on synaptic connectivity, DAT-Cre or Vgat-Cre (Cre/+Slc32a1) mice were injected with AAV1-FLEX-SaCas9-U6-sgNtn1 and AAV1-FLEX–YFP  neurons in DAT-Cre mice injected with Ntn1 CRISPR or control virus, and from non-YFP-expressing (presumptively non-GABA) neurons in Vgat-Cre injected mice. We did not observe significant non-cell autonomous effects on mEPSCs from non-targeted cells  and assayed them in multiple behavioral paradigms. First, we monitored day-night locomotion in control and AAV1-FLEX-SaCas9-sgNtn1 injected DAT-Cre mice. No significant differences were detected  followed by a fixed ratio 5 (FR5) schedule of reinforcement in which one or five lever presses are required to obtain a food reward, respectively. We did not observe significant differences in either of these behavioral tasks  paradigms. Although acoustic startle responses were reduced in AAV1-FLEX-SaCas9-sgNtn1 injected mice, this did not reach significance  into the VTA as described previously and tested these mice using the same behavioral paradigms described above. In contrast to Ntn1 mutagenesis in dopamine neurons, this manipulation in VTA GABA neurons resulted in a significant increase in locomotor activity  compared to controls that remained elevated on the second day of extinction training   , and loscontrol) , and assNtn1 loss of function in VTA GABA and dopamine neurons largely reversed the hyperlocomotor phenotype  and DCC in humans have been associated with several dopamine-associated psychiatric conditions, including neurodevelopmental disorders such as schizophrenia . Mice injected with CRISPR/YFP were allowed 4–5 weeks of recovery after surgery to allow for viral expression, mutagenesis, and protein turnover before any testing. Dat-Cre (Cre/+Slc6a3) mice and Vgat-Cre (Cre/+Slc32a1) mice were obtained from Jackson Laboratories (Strain # 006660 and 028862).All procedures were approved and conducted in accordance with the guidelines of the University of Washington’s Institutional Animal Care and Use Committee (protocol number: 4249–01). Mice were housed on a 12:12 light:dark cycle with All adeno-associated viruses (AAV) for CRISPR/SaCas9 mutagenesis were produced in-house, as previously described . CRISPR All mice used were 8–10 weeks of age at the time of surgery. Mice were inducted using isoflurane at 5.0% and held at 2% throughout the procedure. Mice were stereotaxically injected bilaterally into the VTA using the following coordinates in mm, relative to bregma: A/P: –3.25; M/L±0.5; D/V: (–4.9) – (–4.4), total volume 0.5 µL into each side. A/P coordinates were adjusted for Bregma/Lambda distances using a correction factor of 4.2 mm.Th (Mm-Th), Ntn1 (Mm-Ntn1-C2), and Slc32a1 . Slides were coverslipped with Fluoromount with DAPI (Southern Biotech) and imaged using a confocal fluorescent microscope  and Keyence Fluorescence Microscope (Keyence). Quantification of co-labeled cells was performed using CellProfiler, with thresholding and cell identification/overlap for each channel verified for each image manually prior to quantification.Male and female mice  were used to verify mRNA expression in the VTA using RNAscope. Brains were flash-frozen in 2-methylbutane and representative coronal sections that spanned the VTA were sliced at 20 µm and slide mounted for hybridization. Sections were prepared for hybridization per the manufacturer’s  instructions using probes for Mice were anesthetized with pentobarbital and transcardially perfused with PBS followed by 4% PFA. Brains were post-fixed for 24 hr in PFA at 4°C, followed by 48 hr in 30% sucrose. The VTA was coronally sectioned at 30 µm. Sections were kept in PBS with 0.3% Sodium Azide. Free-floating sections were treated with 0.3% TBS-Triton-X 100 3x10 min, blocked in 3% Normal Donkey Serum for 1 hr and treated overnight in primary antibody. Following 1–3 hr in secondary antibody (JacksonImmuno), sections were slide mounted and coverslipped with Fluoromount with DAPI. Images were collected on a Keyence Fluorescence Microscope (Keyence). For CRISPR validation, male and female DAT-Cre mice (8–12 weeks old) received AAV1-FLEX-SaCas9-U6-sgNtn1/AAV1-FLEX-YFP (Ntn1-cKO) or AAV1-FLEX-SaCas9-U6-sgROSA26/AAV1-FLEX-YFP (controls) injections as described above, and quantification of co-labeled cells for immunohistochemistry were performed using ImageJ 1.53 Cell Counter/Multi-point tool. Total Th-positive cells were recorded for all images and averaged across all slices for each mouse to give a total number of Th-positive cells. Primary antibodies used: mouse anti-TH , chicken anti-Netrin-1 , and rabbit anti-HA .2/CO2. Horizontal (200 µm) brain slices were prepared from 12 to 20 week-old mice in a slush NMDG cutting solution . Slices recovered for ~12 min in the same solution warmed in a 32 °C water bath, then transferred to room temperature HEPES-aCSF solution . Slices recovered for an additional 30–60 min in HEPES solution at room temp. Whole-cell patch-clamp recordings were made using an Axopatch 700B amplifier (Molecular Devices) using 3–5 MΩ electrodes. Recordings were made in aCSF  at 32 °C continually perfused over slices at a rate of ~1 ml/min. VTA dopamine and non-dopamine neurons were identified by fluorescence.Mice injected with CRISPR/YFP were allowed 4–5 weeks of recovery after surgery to allow for viral expression, mutagenesis, and protein turnover. All solutions were continuously bubbled with Osolution  (in mM: A receptor-mediated events. For miniature inhibitory postsynaptic currents (mIPSCs), the internal solution contained: 135 mM KCl, 12 mM NaCl, 0.05 mM EGTA, 100 mM HEPES, 0.2 mM Mg-ATP, 0.02, and Na-GTP mM. To block glutamatergic events, 2 mM kynurenic acid was bath applied in the ACSF. All mIPSCs and mEPSCs cells were recorded in the presence of 1 mM tetrodotoxin (TTX) to block action potentials. Cells were held at −60 mV for a minimum of 5 minprior to data acquisition. Data were analyzed using Clampfit 10.3 .For miniature excitatory postsynaptic currents (mEPSCs), the internal solution contained: 130 mM K-gluconate, 10 mM HEPES, 5 mM NaCl, 1 mM EGTA, 5 mM Mg-ATP, 0.5 mM Na-GTP. Picrotoxin (200 μM) was added to ACSF to block GABAA receptor-mediated events. Electrical stimulation was delivered using a concentric bipolar electrode placed rostral to the VTA. Data were analyzed using Clampfit 10.3 .For PPR, the internal solution contained: 130 mM K-gluconate, 10 mM HEPES, 5 mM NaCl, 1 mM EGTA, 5 mM Mg-ATP, 0.5 mM Na-GTP. Picrotoxin (200 μM) was added to ACSF to block GABAPicrotoxin (100 μM) and tetrodotoxin (500 nM) were included in the bath.For AMPA currents, neurons were held at −60 mV and 50 μM cyclothiazide was perfused onto the slice for 30 s, followed by 1 μM AMPA (with cyclothiazide) for 30 s. For NMDA currents, neurons were held at + 40 mV and 50 μM NMDA was perfused onto the slice for 30 s. Four weeks after surgery, baseline locomotion was measured using locomotion chambers (Columbus instruments) that use infrared beam breaks to calculate ambulatory activity. Mice were singly housed in Allentown cages with reduced corncob bedding and provided with ad libitum access to food and water. Locomotion was monitored continuously for three nights two days.Mice were placed in a large circular arena (120 cm diameter) and activity was recorded for a period of 10 min using Ethovision software. Zones signifying arena edge and center were generated for each video using Ethovision and kept consistent in size and placement across all trials. Time in center, time on edge, and total distance were calculated. Thigmotaxis, the tendency of mice to remain in close proximity to the walls of an enclosure, has been validated as a measure of anxiogenic behavior , with inPrior to testing, all mice were food restricted to 85% body weight and maintained at this level throughout operant conditioning. Mouse weights are included in Acoustic startle responses were measured using acoustic startle chambers (San Diego Instruments). Prior to testing mice received a 10 min habituation period. Background noise was maintained at 65 dB throughout testing. After habituation, mice were presented with 5, 40 ms duration 120 dB, pulse-alone trials to obtain baseline startle responses, followed by 50 trials of either a startle pulse-alone, 1 of 3 prepulse trials, or a null trial, in which no acoustic stimulus is presented. Startle trials consisted of a 40 ms, 120 dB pulse of white noise. The three prepulse trials consisted of a 20 ms prepulse of 70-, 75-, or 80 dB intensity  that preceded 120 dB startle pulse by 100 ms. Peak amplitude of the startle response (65 ms after pulse onset) was used as the measure of startle response magnitude.No sex differences were observed in any of the behavioral or electrophysiological results.Data were analyzed for statistical significance using GraphPad Prism. All statistical tests were two-sided and corrected for multiple comparisons where appropriate. All experiments were repeated at least twice using a double-blind design.All newly created reagents are freely available upon request to the corresponding author. This manuscript reports an important, previously unappreciated, non-developmental role for the guidance cue netrin-1 in midbrain physiology and related behavior in adult animals. Using multiple experimental tools in adult mice, the study convincingly shows that netrin-1 within midbrain dopamine and GABA neurons is necessary to maintain dopamine excitatory tone and plays a role in motivated and anxiety-like behavior. This paper will be of interest to neuroscientists studying dopamine function and/or motivated behavior and those interested in ways that neurodevelopmental genes can continue to play a role in neuronal function and behavior into adulthood. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers opted to remain anonymous.Thank you for submitting the paper "Netrin-1 regulates the balance of glutamatergic connectivity in the adult ventral tegmental area" for consideration by Comments to the Authors:eLife.We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered further for publication by eLife revision .We agreed that the work addresses an interesting question and that the manipulation to knockdown netrin-1 in adult VTA was elegant. However, we noted several concerns. These include unaddressed issues related to the precise role of netrin function in DA neurons leading to challenges in interpreting the behavioral results, the lack of mechanistic insight into the observed effects of glutamate, and contextualization in prior literature examining netrin's effects in the VTA. Addressing the concerns is likely to be well beyond the scope of an Reviewer #1 (Recommendations for the authors):The study seeks to investigate whether the guidance cue netrin-1 in the VTA is involved in the regulation of glutamatergic synaptic connectivity of dopamine and GABA neurons and influences behaviors known to be mediated by mesolimbic dopamine systems.The study involves multiple approaches: viral mediated inactivation of NTn1 in VTA GABA or dopamine neurons, using Cre inducible CRISPR/Cas9; in situ hybridization to confirm colocalization of Ntn1 and tyrosine hydroxylase or Ntn1 and SlC31a1; electrophysiological recordings to evaluate changes in neural excitability of dopamine or GABA neurons, a battery of behavioral tests to evaluate whether loss of Ntn1 in either cell type alters sensorimotor gating function and motivated and anxiety-like behaviors.Major strengths of the paper include (i) the novelty of the question addressed and the multidisciplinary approach, (ii) most of the experiments address functional questions, (iii) the careful characterization of functional and behavioral phenotypes for the majority of the experiments, (iv) the generation of a mechanistic model potentially explaining how netrin-1 in the adult VTA, by regulating the excitatory tone of dopamine and GABA neurons, regulates overall dopamine excitability and behavior.Weaknesses of the study are mainly related to the interpretation of their results and the conclusions made: (i) Regarding the results from the in situ hybridization experiment, the authors do not compare their findings with results from a recent single-cell RNA seq study (PMID: 35385745). There is a discrepancy between the findings, particularly in the proportion of GABA neurons expressing NTn1 . The authors do not mention this issue in their results or discussion. (ii) The authors conclude that their findings demonstrate that netrin-1 regulates the balance of glutamatergic connectivity in the VTA . Indeed they find that downregulation of Ntn1 in VTA dopamine or GABA cells reduces the frequency and amplitude of their miniature excitatory postsynaptic currents (mEPSCs), without altering miniature inhibitory postsynaptic currents (mIPSCs) or leading to statistically reliable changes in frequency or amplitude in other (non-specified) cell types. They also show that reduced mEPSCs is not mediated by changes in presynaptic release, as revealed by paired-pulse ratio measures. However, these findings are not directly linking electrophysiological changes to alterations in glutamatergic synaptic connectivity (iii) Whether NTn1 deletion leads to dopamine or GABA neuronal loss remains unknown. This is important considering previous studies linking (or not) changes in the netrin-1 system in VTA neurons and cell loss. (iv) The study shows that downregulation of Ntn1 in dopamine neurons has no significant effect on reward but influences anxiety-like behaviors. In contrast, downregulation of Ntn1 in GABA neurons produces changes in most of the behaviors tested. It is not clear, however, whether increased locomotor activity in mice with Ntn1 deletion in GABA neurons could influence changes in lever pressing in the operant behaviors. (v) Regarding the changes in extinction training in the mice with reduced Ntn1 in GABA neurons, it seems that they are lever pressing at a higher rate from the beginning but they extinguish their behavior at a similar rate . (vi) the model and hypothesis put forward and tested in the experiments shown in Figure 6 are very interesting. However, the results obtained do not justify the conclusion that Loss of Ntn1 function in both cell types simultaneously largely rescues the consequences induced by GABA- only Ntn1 deletion.To be able to link the electrophysiological changes to glutamatergic synaptic connectivity, other experiments are required, including assessing structural changes (e.g. PMID: 24174661) in dopamine and GABA neurons as well as the proportion of AMPA/NMDA receptors and AMPA/NMDA ratios. In this regard, there is a previous study relating the netrin-1 guidance cue system in adult VTA synaptic plasticity (PMID: 20345916).Whether NTn1 deletion leads to dopamine or GABA neuronal loss could be addressed using stereology.To be able to conclude that the loss of Ntn1 function in both cell types simultaneously largely rescues the consequences induced by GABA- only Ntn1 deletion, the electrophysiological properties of dopamine  neurons could be assessed. This experiment will also test more directly the model the authors are proposing.In the discussion, evidence showing the role of glutamatergic inputs of VTA dopamine neurons on behavior needs to be revised more carefully .Reviewer #2 (Recommendations for the authors):This manuscript by Cline et al. sought to define the role of the axonal guidance cue netrin-1 in synaptic signaling in the ventral tegmental area (VTA). The authors used CRISPR-Cas9 mutagenesis to reduce netrin-1 expression/function in the two predominant neuronal types in the VTA, dopaminergic and GABAergic neurons. This work builds on previous work from this group showing a role for the axon guidance receptor ROBO2 in inhibitory connectivity in adult VTA. Netrin-1 is examined here because of its persistent expression in the VTA into adulthood, despite its established role as a developmental protein. A strong combination of techniques is used, including selective knockdown of the netrin-1 gene in multiple neuron types in the VTA, patch clamp electrophysiology, and several behavioral assays. The results clearly indicate that knockdown of netrin-1 specifically in either GABA or dopamine neurons reduces miniature glutamatergic synaptic currents specifically in that cell type. Interestingly, inhibitory input was not significantly affected. This is consistent with previous reports of effects on excitatory synapses in the hippocampus. Robust behavioral consequences were only reported in the GABA neuron knockdown and were not evident when netrin-1 was knocked down in both cell types. The authors conclude that netrin-1 is important for maintaining the balance of excitatory input onto the two main neuronal subtypes in the VTA. This conclusion is largely supported by the results from the experiments, which were performed rigorously. The manuscript itself was easy to follow and well written, save for some minor omissions.Operant responding for food was used as one of the dependent measures in Figures 4 and 5, with differences observed in the GABA neuron knockdown, however, one omission of the study was that body weights of the mice before and especially after treatment were not reported. It may be that viral knockdown of netrin-1 in one cell type has effects for instance on satiety, producing effects on behavior along with energy balance. The addition of body weight data would help round out the data set and, if different, might help with interpretation. Details were also not provided for the food restriction procedure that was used during operant conditioning for food pellet responding, which could have further interacted with the netrin-1 manipulation in the mouse lines to affect physiology, behavior, or both.As discussed above, most of this study is strong, with interpretations supported by largely convincing results. However, the manuscript could be improved with additions and clarifications.While both cell types showed increased mEPSC frequency and amplitude after netrin-1 knockdown, only the GABA neuron knockdowns showed robust behavioral effects, including increased locomotion (day and night), and increased operant responding for food, and decreased acoustic startle and pre-pulse inhibition. As some of the behavioral tasks involve responding for food pellets, interpretation of the results would benefit from reporting the weights of the mice before and after treatment. Additionally, details about the food restriction procedure that was used for operant conditioning should be provided. Several reports have identified the effects of the feeding state on dopamine neuron excitability and synaptic input to the area. Was the food restriction controlled to a percent loss of body weight, applied acutely or chronically, done throughout the operant study or just during FR1, etc.? Did the mice always eat the pellets when performing the operant task? It's interesting that responding in all of the dopamine mice stayed at the same values when they switched from FR1 to FR5, but in the GABA mice, the control animals fell while the netrin-1 knockdowns stayed the same. Full disclosure of these details would help the reader interpret small observations in the data and later assist in reproducing the results, should they wish to do so.The evidence for the anxiety behavioral phenotype in the dopamine neuron-specific netrin-1 knockdown is pretty thin, as a time-in-center measure in an open field could be affected by other factors, and other supporting data for instance from elevated mazes were not used. Admittedly, anxiety can be difficult to show in mice. However, Reference 20 which was used to support the "proposed role of dopamine in the modulation of anxiety-related behavior" was a dead-end non-citation. If the authors wish to keep this part of their interpretation they should bolster the explanation of the link between dopamine and anxiety with further evidence . Also, details about the meaning of "time in center" and "time on edge" should be provided in the Methods.The extinction result in the GABA mice is presented as "a significant delay in the rate of extinction following reinstatement of FR1." This is hard to interpret because responding immediately before extinction is not given and the data are presented only as raw numbers instead of percent of baseline. If the netrin-1 knockdowns were responding more at FR1 when they were returned to that condition, the shape of the curve would actually indicate no difference in extinction. The same could be true in Figure 6 with the double cell knockdowns. Depending on what the data look like, these graphs may be more accurately presented as normalized numbers. This is a minor issue in the scheme of this paper but it should nonetheless be clarified.The schematics in Figure 6A show that the effects on the GABA neurons proceed through the dopamine neurons. While this is entirely plausible, the authors never actually show this experimentally, for instance by locally manipulating GABA input in virus-injected mice. It may be at least as likely that the important interactions occur in the nucleus accumbens, or some other area to which both cell types project. As a minimum, the authors should point out this caveat in the Discussion, or point out any other possibility that could also explain the somewhat surprising data in Figure 6.There's some confusion about the Ntn1 co-localization in Figure 1. The language in the caption and Figure 1 itself seem clear. However, the text on page 3 seems to contradict the language in the figure caption. The way it is worded, instead of 64, 30, and 6% shouldn't these numbers be 72, 18, and 10%? Please clarify with the correct information, or point out where the confusion lies.In the abstract, "simultaneously" is placed confusingly in the sentence about rescuing the GABA phenotype. This would be more clear if the sentence started "Simultaneous loss… in both cell types."Figure 1D has two  scale bars.Reviewer #3 (Recommendations for the authors):+ or GABA VTA neurons. While the loss of netrin-1 in dopamine neurons did not significantly influence behaviour, loss of netrin-1 in GABAergic neurons increased locomotor activity, effort for rewards, extinction delay, and decreased prepulse inhibition. Finally, effects on locomotor activity, reward seeking, and prepulse inhibition observed in GABA-netrin mice were not present when there was the loss of netrin-1 in both dopamine and GABA neurons, suggesting that loss of netrin-1 in both could restore the behavioural effects of loss of netrin-1 in GABA neurons.In this study, they used genetic strategies to decrease Netrin-1 expression in either dopamine or GABA neurons of the VTA. Reduction of Netrin-1 expression in VTA dopamine neurons decreased excitatory, but not inhibitory postsynaptic currents onto THMajor strengths:This is a nicely written, clearly illustrated study describing the loss of netrin-1 in VTA dopamine neurons and GABA neurons. The authors use an elegant genetic methodology to knock down netrin-1 in select populations of neurons within the VTA. They use a battery of behavioural assays to examine the effects of this knockdown.Major weaknesses:While this study provides a potential physiological mechanism underlying the changed behavioural effects, it doesn't connect these changes to the behaviour or identify the mechanism by which adult netrin-1 influences these changes in synaptic transmission. While netrin-1 is involved in synapse formation during development, it is not clear how netrin-1 is influencing synapses in adulthood. For example, is it necessary for the stabilization of synapses?Author suggestions:The authors propose that loss of netrin-1 in dopamine neurons leads to enhanced excitation and decreased inhibition, whereas loss in GABA neurons would lead to enhanced inhibition and decreased excitation, and the E:I ratio would be balanced when netrin is lost from both cell population. However, they do not test this assertion by measuring excitatory:inhibitory ratio in each model. This would support their hypotheses in figure 6.Some of the discussion focuses on the role of netrin-1 in development. However, the manipulations done in this paper were to remove netrin-1 in adulthood after axon migration and synapse formation occur. They do not discuss what the 'adult function' of netrin-1 is. While it seems to play a role in excitatory synaptic transmission, given its effects on mEPSCs, the authors did not provide sufficient information to conclude if this was a reduction in the number of synapses, a silencing of synapses, or a decrease in release probability with compensatory postsynaptic changes. Experiments addressing how adult netrin-1 signaling in the VTA specifically influences synaptic transmission onto dopamine or GABA neurons of the VTA may highlight how netrin-1 might be contributing to associated behavioral changes.eLife. Your revised article has been evaluated by Kate Wassum (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Netrin-1 regulates the balance of glutamatergic connectivity in the adult ventral tegmental area" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:Essential revisions:We have a few remaining concerns that can be addressed with an additional analysis of existing data (see R3 point #1) and edits to the text to add clarity, change language, temper conclusions, discuss alternatives, etc.Please provide a point x point response to each reviewer point along with your revision.Reviewer #1 (Recommendations for the authors):This resubmitted manuscript is substantially improved from the previous version. Additions and clarification to methods and interpretations have largely addressed my previous concerns, which were minor. Additional discussion has bolstered the notion that netrin-1 is impotant for maintaining the balance of excitatory and inhibitory inputs to dopamine neurons. The addition of maze data would have bolstered conclusions about the anxiety phenotype, but "time on edge" and locomotor data was added and this was always a minor point that did not detract substantially from the rest of the manuscript.One item does need to be clarified. The experiment represented in Figure 3 Supplement 3 was performed to address a concern of Reviewer 1, however the text describing this is very confusing. The Results describe these data as evoked EPSCs following bath perfusion of AMPA or NMDA. The data themselves seem to be showing changes in holding current in a voltage clamp experiment, although the axis labels and the lack of sample traces leave this in doubt. If it is holding current, more details should to be provided in the figure . If instead it is evoked EPSCs in the presence of bath perfused agonists, then other details also need to be provided to make this result make sense (such as sample traces and an explanation of what they were after).The addition of body weight data and food restriction information is appreciated. However in the Response to Reviewers the level of restriction was listed as 85% of initial body weight, whereas in the manuscript this is given as 80%. Please make sure the correct number appears in the manuscript.Reviewer #2 (Recommendations for the authors):The revised version improved the interpretation of the results, and I am pleased that the authors replied to most of my comments on the previous version. They added needed citations, clarification on their manipulation, and additional experiments to understand the changes in synaptic connectivity better and clarify the electrophysiological changes that occur with Ntn1 mutagenesis, as well as their proposed model.The following issues remain unresolved:– Since the study does not assess alterations in neuronal structure and connectivity, we suggest the word "connectivity" to be dropped or modified.+ or GABA+ cells in the mice with conditional netrin-1 KO. The statement "Total Th-positive cells were recorded for all images and averaged across all slices for each mouse to give a total number of Th-positive cells" needs to be revised, because total number of cells can only be assessed using stereological analysis. It remains unknown whether NTn1 deletion leads to dopamine or GABA neuronal loss. This is an important issue that needs to be acknowledged in the manuscript. Please see– It appears that stereology was not performed to calculate the number of neurons expressing netrin-1 in THhttps://www.embopress.org/doi/full/10.15252/embj.2020105537– Regarding the increase in locomotor activity observed after the downregulation of Ntn1 in GABA neurons, the authors argue that the level of responding in the FR1 task is not altered in GABA-Ntn1 mutant mice, suggesting that elevated responding in the FR5 and PR assays is not likely a reflection of hyperactivity. Yet, increasing the response rate in the FR1 protocol is no possible. Once the animals respond to the reward in the FR1, the lever retracts, and no more lever-pressing is possible until a reward is collected. The statement added by the authors "however, we did not observe differences in operant responding during FR1 training or reinstatement indicating that the observed effects are likely a reflection of altered motivational states rather than general hyperactivity" needs to be modified because hyperactivity may be influencing the findings reported– Regarding the lever pressing extinction data, (1) the authors acknowledge that mice with Ntn1 cKO in GABA VTA neurons have similar extinction rates than WT. They now need to mention that this finding suggests that the increase in the number of lever-pressing during the extinction phase in Ntn1 cKO in GABA mice is most likely associated with an increase in baseline responding rather than with extinction deficits. This same point needs to be highlighted for the Ntn1 cKO in DATIRES::Vgat-Cre mice too. (2) the rate of extinction is not analyzed nor discussed. (3) it is unclear how plotting the extinction bin data for day1 and day 2 and for day 4 and day 5, for suppl Figures5 and 6 respectively, addresses this issue. The authors need to consider removing these data because they do not seem to provide relevant information.Reviewer #3 (Recommendations for the authors):I appreciate the addition of the bath application of AMPA or NMDA experiments. While these add support to the effect of netrin being postsynaptic, it should be indicated that these experiments do not distinguish between synaptic responses and extrasynaptic responses. Further a change in holding current after bath application of AMPA could be due to reduced synapse number that is associated with a reduced postsynaptic AMPA receptor complement. Further, while they show no change in PPR consistent with no effect on release probability (Pr), their decrease in mEPSC amplitude and frequency could be consistent with a reduction in synapse number (number of release sites N) or a change in number or function of postsynaptic AMPA receptors. To distinguish this, you can measure 1/CV2 which depends on N and Pr but is independent of quantal size. Given that there is no change in Pr, if you see a change in 1/CV2 it might be consistent with a decrease in release sites as opposed to a change in postsynaptic receptor number or function. Alternatively, if there is no change in 1/CV2, you can make a stronger conclusion that the netrin manipulation is altering number or function of postsynaptic AMPA receptors.Regarding the shift in the E:I balance, while I do not disagree with their model, I was proposing that they could test their model directly by recording mIPSCs at the reversal potential for EPSCs (0 to 10mV) and mEPSCs at the reversal potential for GABA mIPSCs (~-60 to -70mV) and converting the average peak current amplitude into conductance. This way, you can directly measure the amount of inhibitory current and amount of excitatory current the same cell is receiving. Perhaps this experiment is beyond the scope of the current manuscript, but when discussing their model, they could indicate that they could test the validity of this hypothesis in future experiments. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1 (Recommendations for the authors):The study seeks to investigate whether the guidance cue netrin-1 in the VTA is involved in the regulation of glutamatergic synaptic connectivity of dopamine and GABA neurons and influences behaviors known to be mediated by mesolimbic dopamine systems.The study involves multiple approaches: viral mediated inactivation of NTn1 in VTA GABA or dopamine neurons, using Cre inducible CRISPR/Cas9; in situ hybridization to confirm colocalization of Ntn1 and tyrosine hydroxylase or Ntn1 and SlC31a1; electrophysiological recordings to evaluate changes in neural excitability of dopamine or GABA neurons, a battery of behavioral tests to evaluate whether loss of Ntn1 in either cell type alters sensorimotor gating function and motivated and anxiety-like behaviors.Major strengths of the paper include (i) the novelty of the question addressed and the multidisciplinary approach, (ii) most of the experiments address functional questions, (iii) the careful characterization of functional and behavioral phenotypes for the majority of the experiments, (iv) the generation of a mechanistic model potentially explaining how netrin-1 in the adult VTA, by regulating the excitatory tone of dopamine and GABA neurons, regulates overall dopamine excitability and behavior.Weaknesses of the study are mainly related to the interpretation of their results and the conclusions made: (i) Regarding the results from the in situ hybridization experiment, the authors do not compare their findings with results from a recent single-cell RNA seq study (PMID: 35385745). There is a discrepancy between the findings, particularly in the proportion of GABA neurons expressing NTn1 . The authors do not mention this issue in their results or discussion.Ntn1. We have also performed snRNA seq in mouse VTA and find that netrin 1 displays partially enriched expression in one of 3 of the major GABA populations that we identified (unpublished data). Differences in the proportion of GABA neurons we identified that express Ntn1 compared to the data in PMID: 35385745 may reflect differences between rat and mouse, or increased sensitivity of in situ hybridization compared to snRNA seq. We have added discussion of this point to the updated manuscript. The role of Ntn1 expression in glial cells is interesting, and we have added this point to our revised discussion.We find that approximately 30% of Vgat-expressing neurons also express Ntn1. Analysis of data from PMID: 35385745 confirms that a subset of neurons in their population designated GABA1, express (ii) The authors conclude that their findings demonstrate that netrin-1 regulates the balance of glutamatergic connectivity in the VTA . Indeed they find that downregulation of Ntn1 in VTA dopamine or GABA cells reduces the frequency and amplitude of their miniature excitatory postsynaptic currents (mEPSCs), without altering miniature inhibitory postsynaptic currents (mIPSCs) or leading to statistically reliable changes in frequency or amplitude in other (non-specified) cell types. They also show that reduced mEPSCs is not mediated by changes in presynaptic release, as revealed by paired-pulse ratio measures. However, these findings are not directly linking electrophysiological changes to alterations in glutamatergic synaptic connectivity.Ntn1 mutagenesis . We believe this addition clarifies the electrophysiological changes that occur with Ntn1 mutagenesis.We apologize for our confusion, but we are not quite clear what the reviewers means when they state the mEPSCs are “not directly linking electrophysiological changes to alterations in glutamatergic synaptic connectivity”. mEPSC and mIPSP frequency and amplitude have been the gold standard in establishing excitatory synaptic connectivity since the original description of quantal synaptic transmission at the motor endplate , the introduction of TTX to block action potential firing  and the isolation of miniature inhibitory synaptic currents . A reduction in mEPSC frequency with no change in paired pulse ratio is commonly interpreted as a loss of functional synapses. Additional measures of synaptic connectivity, such as ultrastructural analysis can be performed, but dopamine neurons of the ventral midbrain have long been viewed as generally aspiny neurons , though there is certainly no clear consensus on this . To better resolve the observed changes in synaptic connectivity in mEPSCs, we included bath application of AMPA and NMDA in the updated manuscript to quantify the degree of ‘total’ receptors present on surface of these cells with or without (iii) Whether NTn1 deletion leads to dopamine or GABA neuronal loss remains unknown. This is important considering previous studies linking (or not) changes in the netrin-1 system in VTA neurons and cell loss.Ntn1, which are more easily assessed by IHC, and determined that loss of Ntn1 function does not appear to result in the loss of dopamine neurons within the VTA.We agree that determining if Ntn1 deletion leads to neuronal loss is an important question. To address this, we have counted TH-positive cells in the VTA of mice with mutagenesis of (iv) The study shows that downregulation of Ntn1 in dopamine neurons has no significant effect on reward but influences anxiety-like behaviors. In contrast, downregulation of Ntn1 in GABA neurons produces changes in most of the behaviors tested. It is not clear, however, whether increased locomotor activity in mice with Ntn1 deletion in GABA neurons could influence changes in lever pressing in the operant behaviors.We agree that locomotor effects can confound interpretations in behavioral assays. It is important to note that the level of responding in the FR1 task was not altered in GABA-Ntn1 mutant mice, suggesting that elevated responding in the FR5 and PR assays are not likely a simple reflection of hyperactivity. We have more explicitly addressed this in the revised manuscript.(v) Regarding the changes in extinction training in the mice with reduced Ntn1 in GABA neurons, it seems that they are lever pressing at a higher rate from the beginning but they extinguish their behavior at a similar rate .The reviewer makes an interesting point. The extinction burst observed in controls is part of the extinction process that reflects the invigoration of the behavioral response when the outcome does not match expectation at the beginning of the extinction process. We have altered the manuscript to be more specific on what we are referring to regarding the deficits in extinction behavior and have provided additional analysis of the rate of responding on the days of extinction training where differences were observed in the overall numbers of lever presses but not the slopes of the curves.(vi) the model and hypothesis put forward and tested in the experiments shown in Figure 6 are very interesting. However, the results obtained do not justify the conclusion that Loss of Ntn1 function in both cell types simultaneously largely rescues the consequences induced by GABA- only Ntn1 deletion.ELife), or blocking selectively GABA release from VTA GABA neurons  results in hyperactivity and increased operant responding, though to a much greater degree than the effects observed here. We, and others have also shown that loss of glutamate signaling in dopamine neurons has only a modest behavioral effect , so it is not surprising that reducing mEPSCs onto VTA dopamine neurons has little effect in this context. The partial rescue of the GABA-Ntn1 knockout experiment suggests that reducing glutamatergic input onto dopamine neurons can abrogate effects associated with reduced inhibitory tone, thus restoring the excitatory and inhibitory balance. In the revised discussion we outline a detailed mechanism that we propose explains this observation.We apologize for the lack of clarity. We have altered the discussion to more explicitly provide an explanation of how mutagenesis in both cell types can result in a diminution of the observed phenotype in the GABA-Ntn1 knockout mice. In the model proposed, reduced excitatory input onto GABA neurons results in a reduced inhibition of dopamine neurons causing the observed phenotypes. For reference, we have previously shown that blocking all synaptic transmission from GABA neurons in the VTA  in dopamine and GABA neurons as well as the proportion of AMPA/NMDA receptors and AMPA/NMDA ratios. In this regard, there is a previous study relating the netrin-1 guidance cue system in adult VTA synaptic plasticity (PMID: 20345916).Ntn1 mutant dopamine neurons. We have also made sure that we reference the above highlighted paper which describes the impact of Dcc heterozygous loss of function on amphetamine sensitization.An initial address of this point was provided above. Assessing AMAPA/NMDA ratio is another measure of synaptic strength; however, this assumes that NMDA receptor levels remain unchanged, which we know from previous studies is not always true in the VTA . To address this, we recorded both AMPA and NMDA-evoked current in control and Whether NTn1 deletion leads to dopamine or GABA neuronal loss could be addressed using stereology.Ntn1 deletion did not result in a loss of dopamine neurons.We have included a count of the number of virally labelled dopamine neurons to address this point and show that To be able to conclude that the loss of Ntn1 function in both cell types simultaneously largely rescues the consequences induced by GABA- only Ntn1 deletion, the electrophysiological properties of dopamine  neurons could be assessed. This experiment will also test more directly the model the authors are proposing.Ntn1 loss of function are and how they are consistent with what is known about the organization of the VTA cell types.This experiment is more difficult to address than the reviewer may appreciate. Both dopamine and GABA neurons express Cre, so fluorescent isolation of these cells will not be possible. Ih currents can be used, but not all dopamine neurons are Ih positive. This is particularly problematic as the number of excitatory synapses onto GABA neurons is higher than onto dopamine neurons, thus there will be a large spread in the distribution of responses making statistical analysis difficult to achieve. To address this, we have been more explicit in our discussion of what we hypothesize the double mutagenesis is achieving and why this is important for considerations of the cell-type and circuit-specific impacts of In the discussion, evidence showing the role of glutamatergic inputs of VTA dopamine neurons on behavior needs to be revised more carefully .We have expanded this area of the discussion to address this point.Reviewer #2 (Recommendations for the authors):This manuscript by Cline et al. sought to define the role of the axonal guidance cue netrin-1 in synaptic signaling in the ventral tegmental area (VTA). The authors used CRISPR-Cas9 mutagenesis to reduce netrin-1 expression/function in the two predominant neuronal types in the VTA, dopaminergic and GABAergic neurons. This work builds on previous work from this group showing a role for the axon guidance receptor ROBO2 in inhibitory connectivity in adult VTA. Netrin-1 is examined here because of its persistent expression in the VTA into adulthood, despite its established role as a developmental protein. A strong combination of techniques is used, including selective knockdown of the netrin-1 gene in multiple neuron types in the VTA, patch clamp electrophysiology, and several behavioral assays. The results clearly indicate that knockdown of netrin-1 specifically in either GABA or dopamine neurons reduces miniature glutamatergic synaptic currents specifically in that cell type. Interestingly, inhibitory input was not significantly affected. This is consistent with previous reports of effects on excitatory synapses in the hippocampus. Robust behavioral consequences were only reported in the GABA neuron knockdown and were not evident when netrin-1 was knocked down in both cell types. The authors conclude that netrin-1 is important for maintaining the balance of excitatory input onto the two main neuronal subtypes in the VTA. This conclusion is largely supported by the results from the experiments, which were performed rigorously. The manuscript itself was easy to follow and well written, save for some minor omissions.Operant responding for food was used as one of the dependent measures in Figures 4 and 5, with differences observed in the GABA neuron knockdown, however, one omission of the study was that body weights of the mice before and especially after treatment were not reported. It may be that viral knockdown of netrin-1 in one cell type has effects for instance on satiety, producing effects on behavior along with energy balance. The addition of body weight data would help round out the data set and, if different, might help with interpretation.This is an excellent point. All mice were food restricted to 85% of their body weight, and the methods have been changed to reflect that omission. Initial bodyweights at the start of calorie restriction have also been included in the supplemental data.Details were also not provided for the food restriction procedure that was used during operant conditioning for food pellet responding, which could have further interacted with the netrin-1 manipulation in the mouse lines to affect physiology, behavior, or both.We apologize for the oversight and have corrected the methods to reflect the calorie restriction used.As discussed above, most of this study is strong, with interpretations supported by largely convincing results. However, the manuscript could be improved with additions and clarifications.While both cell types showed increased mEPSC frequency and amplitude after netrin-1 knockdown, only the GABA neuron knockdowns showed robust behavioral effects, including increased locomotion (day and night), and increased operant responding for food, and decreased acoustic startle and pre-pulse inhibition. As some of the behavioral tasks involve responding for food pellets, interpretation of the results would benefit from reporting the weights of the mice before and after treatment. Additionally, details about the food restriction procedure that was used for operant conditioning should be provided. Several reports have identified the effects of the feeding state on dopamine neuron excitability and synaptic input to the area. Was the food restriction controlled to a percent loss of body weight, applied acutely or chronically, done throughout the operant study or just during FR1, etc.? Did the mice always eat the pellets when performing the operant task? It's interesting that responding in all of the dopamine mice stayed at the same values when they switched from FR1 to FR5, but in the GABA mice, the control animals fell while the netrin-1 knockdowns stayed the same. Full disclosure of these details would help the reader interpret small observations in the data and later assist in reproducing the results, should they wish to do so.We apologize for the oversight and have corrected the methods to reflect the food restriction used. We have also included the body weights of the animals prior to calorie restriction.The evidence for the anxiety behavioral phenotype in the dopamine neuron-specific netrin-1 knockdown is pretty thin, as a time-in-center measure in an open field could be affected by other factors, and other supporting data for instance from elevated mazes were not used. Admittedly, anxiety can be difficult to show in mice. However, Reference 20 which was used to support the "proposed role of dopamine in the modulation of anxiety-related behavior" was a dead-end non-citation. If the authors wish to keep this part of their interpretation they should bolster the explanation of the link between dopamine and anxiety with further evidence . Also, details about the meaning of "time in center" and "time on edge" should be provided in the Methods.Arch Iran Med. 2015;18(9):591-603.” The reference has been corrected.We apologize for the broken citation. The citation should have read ” Zarrindast MR, Khakpai F. The Modulatory Role of Dopamine in Anxiety-like Behavior. We have also provided additional information re: time in center and time on edge in the methods to add clarification.The extinction result in the GABA mice is presented as "a significant delay in the rate of extinction following reinstatement of FR1." This is hard to interpret because responding immediately before extinction is not given and the data are presented only as raw numbers instead of percent of baseline. If the netrin-1 knockdowns were responding more at FR1 when they were returned to that condition, the shape of the curve would actually indicate no difference in extinction. The same could be true in Figure 6 with the double cell knockdowns. Depending on what the data look like, these graphs may be more accurately presented as normalized numbers. This is a minor issue in the scheme of this paper but it should nonetheless be clarified.We have provided the data on the reinstatement training and have performed additional analysis of the rates of extinction for each of the mice within a given session to more accurately address this concern.The schematics in Figure 6A show that the effects on the GABA neurons proceed through the dopamine neurons. While this is entirely plausible, the authors never actually show this experimentally, for instance by locally manipulating GABA input in virus-injected mice. It may be at least as likely that the important interactions occur in the nucleus accumbens, or some other area to which both cell types project. As a minimum, the authors should point out this caveat in the Discussion, or point out any other possibility that could also explain the somewhat surprising data in Figure 6.We agree that the projection of a subset of GABA neurons to other brain regions may play an important role in the observed phenotypes. We have clarified this in the discussion.There's some confusion about the Ntn1 co-localization in Figure 1. The language in the caption and Figure 1 itself seem clear. However, the text on page 3 seems to contradict the language in the figure caption. The way it is worded, instead of 64, 30, and 6% shouldn't these numbers be 72, 18, and 10%? Please clarify with the correct information, or point out where the confusion lies.We apologize for the confusion and have clarified this in our revised manuscript.In the abstract, "simultaneously" is placed confusingly in the sentence about rescuing the GABA phenotype. This would be more clear if the sentence started "Simultaneous loss… in both cell types."We agree and have made this change.Figure 1D has two  scale bars.We have corrected this inadvertent duplication.Reviewer #3 (Recommendations for the authors):+ or GABA VTA neurons. While the loss of netrin-1 in dopamine neurons did not significantly influence behaviour, loss of netrin-1 in GABAergic neurons increased locomotor activity, effort for rewards, extinction delay, and decreased prepulse inhibition. Finally, effects on locomotor activity, reward seeking, and prepulse inhibition observed in GABA-netrin mice were not present when there was the loss of netrin-1 in both dopamine and GABA neurons, suggesting that loss of netrin-1 in both could restore the behavioural effects of loss of netrin-1 in GABA neurons.In this study, they used genetic strategies to decrease Netrin-1 expression in either dopamine or GABA neurons of the VTA. Reduction of Netrin-1 expression in VTA dopamine neurons decreased excitatory, but not inhibitory postsynaptic currents onto THMajor strengths:This is a nicely written, clearly illustrated study describing the loss of netrin-1 in VTA dopamine neurons and GABA neurons. The authors use an elegant genetic methodology to knock down netrin-1 in select populations of neurons within the VTA. They use a battery of behavioural assays to examine the effects of this knockdown.Major weaknesses:While this study provides a potential physiological mechanism underlying the changed behavioural effects, it doesn't connect these changes to the behaviour or identify the mechanism by which adult netrin-1 influences these changes in synaptic transmission. While netrin-1 is involved in synapse formation during development, it is not clear how netrin-1 is influencing synapses in adulthood. For example, is it necessary for the stabilization of synapses?We agree with the reviewer and have received a similar concern from reviewer 1. A response to this is provided above and we will summarize our experimental approach below.Author suggestions:The authors propose that loss of netrin-1 in dopamine neurons leads to enhanced excitation and decreased inhibition, whereas loss in GABA neurons would lead to enhanced inhibition and decreased excitation, and the E:I ratio would be balanced when netrin is lost from both cell population. However, they do not test this assertion by measuring excitatory:inhibitory ratio in each model. This would support their hypotheses in figure 6.Ntn1 function in either dopamine neurons or GABA neurons results in a loss of excitatory synaptic connectivity with no loss in inhibitory synaptic connectivity. Loss of excitatory input with no change in inhibitory input will bias towards inhibition in the E:I balance. When this occurs in GABA neurons, because local GABA potently inhibits dopamine neurons, the loss of excitation onto GABA neurons biases towards greater inhibition of these cells which disinhibits the dopamine neurons causing a hyperdopaminergic phenotype. We have provided a detailed discussion of the potential mechanism by which loss of Ntn1 in both cell types would restore the E:I balance.We thank the reviewer for their comment. We conclude that loss of Some of the discussion focuses on the role of netrin-1 in development. However, the manipulations done in this paper were to remove netrin-1 in adulthood after axon migration and synapse formation occur. They do not discuss what the 'adult function' of netrin-1 is. While it seems to play a role in excitatory synaptic transmission, given its effects on mEPSCs, the authors did not provide sufficient information to conclude if this was a reduction in the number of synapses, a silencing of synapses, or a decrease in release probability with compensatory postsynaptic changes. Experiments addressing how adult netrin-1 signaling in the VTA specifically influences synaptic transmission onto dopamine or GABA neurons of the VTA may highlight how netrin-1 might be contributing to associated behavioral changes.Ntn1 pointing to a postsynaptic effect.As mentioned above, we have included recordings of AMPA and NMDA receptor mediated currents (via bath application of the specific agonists) in these cells to quantify the levels of these receptors. Our paired-pulse ratio experiments indicate that presynaptic release probability is not altered with the loss of [Editors’ note: what follows is the authors’ response to the second round of review.]Essential revisions:We have a few remaining concerns that can be addressed with an additional analysis of existing data (see R3 point #1) and edits to the text to add clarity, change language, temper conclusions, discuss alternatives, etc.Please provide a point x point response to each reviewer point along with your revision.Reviewer #1 (Recommendations for the authors):This resubmitted manuscript is substantially improved from the previous version. Additions and clarification to methods and interpretations have largely addressed my previous concerns, which were minor. Additional discussion has bolstered the notion that netrin-1 is impotant for maintaining the balance of excitatory and inhibitory inputs to dopamine neurons. The addition of maze data would have bolstered conclusions about the anxiety phenotype, but "time on edge" and locomotor data was added and this was always a minor point that did not detract substantially from the rest of the manuscript.One item does need to be clarified. The experiment represented in Figure 3 Supplement 3 was performed to address a concern of Reviewer 1, however the text describing this is very confusing. The Results describe these data as evoked EPSCs following bath perfusion of AMPA or NMDA. The data themselves seem to be showing changes in holding current in a voltage clamp experiment, although the axis labels and the lack of sample traces leave this in doubt. If it is holding current, more details should to be provided in the figure . If instead it is evoked EPSCs in the presence of bath perfused agonists, then other details also need to be provided to make this result make sense (such as sample traces and an explanation of what they were after).We apologize for the confusion and lack of clarity regarding these experiments. These data were from a voltage clamp experiment in which agonist was bath applied for 30 seconds, and holding current was recorded. The figure legends and axis have been amended to reduce confusion, and the experiment has been better clarified in the Results section.The addition of body weight data and food restriction information is appreciated. However in the Response to Reviewers the level of restriction was listed as 85% of initial body weight, whereas in the manuscript this is given as 80%. Please make sure the correct number appears in the manuscript.The addition of body weight data and food restriction information is appreciated.However in the Response to Reviewers the level of restriction was listed as 85% of initial body weight, whereas in the manuscript this is given as 80%. Please make sure the correct number appears in the manuscript.Reviewer #2 (Recommendations for the authors):The revised version improved the interpretation of the results, and I am pleased that the authors replied to most of my comments on the previous version. They added needed citations, clarification on their manipulation, and additional experiments to understand the changes in synaptic connectivity better and clarify the electrophysiological changes that occur with Ntn1 mutagenesis, as well as their proposed model.The following issues remain unresolved:– Since the study does not assess alterations in neuronal structure and connectivity, we suggest the word "connectivity" to be dropped or modified.We thank the reviewer for their comment. Although we disagree that the measurement of miniature inhibitory and excitatory postsynaptic currents is not a direct measure of synaptic connectivity, we have changed the title to “Netrin-1 regulates the balance of synaptic glutamate signaling in the adult ventral tegmental area”.+ or GABA+ cells in the mice with conditional netrin-1 KO. The statement "Total Th-positive cells were recorded for all images and averaged across all slices for each mouse to give a total number of Th-positive cells" needs to be revised, because total number of cells can only be assessed using stereological analysis. It remains unknown whether NTn1 deletion leads to dopamine or GABA neuronal loss. This is an important issue that needs to be acknowledged in the manuscript. Please see– It appears that stereology was not performed to calculate the number of neurons expressing netrin-1 in THhttps://www.embopress.org/doi/full/10.15252/embj.2020105537Ntn1 deletion the average number of TH+ cells per slice was not statistically different in DAT-Cre Ntn1 cKO mice compared to controls (control: 178.2±12.68 and Ntn1 cKO 173.3±10.75). Although this result is consistent with Ntn1 inactivation not compromising cell viability, without a complete stereological analysis of every neuron within the VTA, we cannot definitively conclude that some cell loss did not occur.”We thank the reviewer for this comment. We have addressed this with the following statement in the Results section: “Following – Regarding the increase in locomotor activity observed after the downregulation of Ntn1 in GABA neurons, the authors argue that the level of responding in the FR1 task is not altered in GABA-Ntn1 mutant mice, suggesting that elevated responding in the FR5 and PR assays is not likely a reflection of hyperactivity. Yet, increasing the response rate in the FR1 protocol is no possible. Once the animals respond to the reward in the FR1, the lever retracts, and no more lever-pressing is possible until a reward is collected. The statement added by the authors "however, we did not observe differences in operant responding during FR1 training or reinstatement indicating that the observed effects are likely a reflection of altered motivational states rather than general hyperactivity" needs to be modified because hyperactivity may be influencing the findings reportedNtn1 cKO mice contributes to the elevated lever press rates during FR5, PR, and extinction.We appreciate this suggestion and have modified the phrasing in the manuscript in response. The phrasing has been changed to: While these data likely reflect altered motivational state with loss of Ntn1, it is also possible that the hyperactivity observed in Vgat-Cre – Regarding the lever pressing extinction data, (1) the authors acknowledge that mice with Ntn1 cKO in GABA VTA neurons have similar extinction rates than WT. They now need to mention that this finding suggests that the increase in the number of lever-pressing during the extinction phase in Ntn1 cKO in GABA mice is most likely associated with an increase in baseline responding rather than with extinction deficits. This same point needs to be highlighted for the Ntn1 cKO in DATIRES::Vgat-Cre mice too. (2) the rate of extinction is not analyzed nor discussed. (3) it is unclear how plotting the extinction bin data for day1 and day 2 and for day 4 and day 5, for suppl Figures5 and 6 respectively, addresses this issue. The authors need to consider removing these data because they do not seem to provide relevant information.Ntn1 mutant mice.We thank the reviewer for this comment and we believe that this is addressed in the response above. With regard to the inclusion of the additional extinction analysis previously requested by the reviewer, we believe that this data provides additional information for the reader to better understand the extinction responding in control and Reviewer #3 (Recommendations for the authors):I appreciate the addition of the bath application of AMPA or NMDA experiments. While these add support to the effect of netrin being postsynaptic, it should be indicated that these experiments do not distinguish between synaptic responses and extrasynaptic responses. Further a change in holding current after bath application of AMPA could be due to reduced synapse number that is associated with a reduced postsynaptic AMPA receptor complement. Further, while they show no change in PPR consistent with no effect on release probability (Pr), their decrease in mEPSC amplitude and frequency could be consistent with a reduction in synapse number (number of release sites N) or a change in number or function of postsynaptic AMPA receptors. To distinguish this, you can measure 1/CV2 which depends on N and Pr but is independent of quantal size. Given that there is no change in Pr, if you see a change in 1/CV2 it might be consistent with a decrease in release sites as opposed to a change in postsynaptic receptor number or function. Alternatively, if there is no change in 1/CV2, you can make a stronger conclusion that the netrin manipulation is altering number or function of postsynaptic AMPA receptors.2 for mEPSCs based on this suggestion and have presented that data in the results and as figure 3 supplemental 3.This is an excellent suggestion. We calculated 1/CVRegarding the shift in the E:I balance, while I do not disagree with their model, I was proposing that they could test their model directly by recording mIPSCs at the reversal potential for EPSCs (0 to 10mV) and mEPSCs at the reversal potential for GABA mIPSCs (~-60 to -70mV) and converting the average peak current amplitude into conductance. This way, you can directly measure the amount of inhibitory current and amount of excitatory current the same cell is receiving. Perhaps this experiment is beyond the scope of the current manuscript, but when discussing their model, they could indicate that they could test the validity of this hypothesis in future experiments.We appreciated the suggestion and do agree that this experiment is beyond the scope of the current manuscript, but we have included this as a suggestion for future experiments in the discussion of this paper.


We find that, once anodic hydrocarbon-to-oxygenate conversion is developed with high selectivities, greenhouse gas emissions associated with fossil-based NH3 and oxygenates manufacture can be reduced by up to 88%. We report that low-carbon electricity is not mandatory to enable a net reduction in greenhouse gas emissions: global chemical industry emissions can be reduced by up to 39% even with electricity having the carbon footprint per MWh available in the United States or China today. We conclude with considerations and recommendations for researchers who wish to embark on this research direction.Chemicals manufacture is among the top greenhouse gas contributors. More than half of the associated emissions are attributable to the sum of ammonia plus oxygenates such as methanol, ethylene glycol and terephthalic acid. Here we explore the impact of electrolyzer systems that couple electrically-powered anodic hydrocarbon-to-oxygenate conversion with cathodic H 2 emissions of the chemicals industry. Coupled electrification of these processes has the potential to reduce total emissions by up to 39% even when using the electricity mix available today.The production of hydrogen, and the refining of hydrocarbons, are significant contributors to the CO Of these emissions of chemicals, 85% arise from the large consumption of fossil-based energy and feedstocks, while 15% are direct emissions from the imperfect selectivity of present-day thermochemical production methods: a significant proportion of the hydrocarbon feedstock is oxidized all the way to carbon dioxide (CO2) instead of to the desired, partially-oxidized product2. Thus, to turn fully carbon-neutral, it is not sufficient simply to switch the energy source from fossil fuels to renewable energy: today’s processes must be replaced by alternatives that do not oxidize hydrocarbons all the way to CO2.Chemicals manufacture is among the top greenhouse gas (GHG) contributors, at 18% of global industrial emissions3) plus oxygenates such as methanol, ethylene oxide, ethylene glycol, propylene oxide, phenol, and terephthalic acid, which contain oxygen as part of their chemical structure2. Dedicated research on new processes to manufacture these chemicals can therefore achieve major impact in reducing net GHG emissions.Considering all chemicals, more than 50% of GHG emissions are attributable to the sum of ammonia  feedstock —less than 2% of the global H2 demand is fulfilled this way5. This is in part because the oxygen evolution reaction (OER) at the anode of water electrolyzers adds considerably to the energy cost of H2 + O26. Today’s state-of-art water electrolyzers consume about 180 MJ of energy per kg H27, which is significantly higher than its lower heating value (LHV) of 120 MJ per kg H2. With present-day price of electricity, water electrolysis is therefore more expensive than steam methane reforming7. Water electrolysis needs to be powered using low-carbon electricity to achieve a net reduction in GHG emissions. Such electricity is still limited in most industrialized countries today, as electricity from renewable sources such as solar and wind are intermittent and not yet widely available. We focused therefore on strategies that take into account the high energy demand of water electrolysis, and that seek to reduce GHG emissions without relying on ~100%-renewable electricity.Despite the existence of a cleaner alternative—water electrolysis to H2 evolution reaction (HER) from water under ambient conditions. We find that by redirecting the energy that would otherwise be consumed in low-value O2 evolution reaction (OER), and instead synthesizing higher-value oxygenates, the approach maximizes the utilization of electricity in producing valuable chemicals. We evaluate, through a prospective life cycle assessment (LCA), the GHG reduction potential compared to thermocatalytic processes. This strategy differs from some prior LCA works which have focused on carbon capture and utilization technologies to reduce emissions associated with the chemicals industry by closing the carbon cycle11. The present LCA model provides a lower bound for carbon emissions showcasing the best-case scenario when the coupled electrolyzer systems are implemented. It can be seen that, once anodic hydrocarbon-to-oxygenate conversion is developed to target higher selectivities, GHG emissions associated with fossil-based NH3 and oxygenates manufacture can be reduced by up to 88%. Low-carbon electricity is not mandatory to enable a net reduction in GHG emissions: indeed, we find that global chemical industry emissions could be reduced by up to 39% even with electricity of carbon intensities (defined as the carbon footprint per MWh electricity) available in the United States and China today12. Taking into account regional production facilities, ~18% of global chemical sites currently conduct both NH3 manufacture and partial oxidations of hydrocarbons to oxygenates at the same site13. These sites can be targeted for implementation of coupled electrolyzer technologies. We close by discussing the gap between industrial needs and present-day electrochemical studies, as well as how the research community can develop electrocatalysts and reaction conditions to realize these new electrolyzer technologies.Here we propose that the electrification of hydrocarbon-to-oxygenate conversions can make a significant impact in reducing the carbon footprint of the chemicals industry, due to the high production volumes and GHG emissions. The main intent of the present paper is to evaluate electrolyzer systems that couple anodic hydrocarbon-to-oxygenate conversion with the cathodic H9. We therefore use the result as a proxy for the chemical industry. The calculated cradle-to-gate GHG emissions include emissions from the provision of feedstock , thermal energy , electricity  as well as direct emissions and waste treatment .We evaluate the global climate impact of chemicals manufacture using an LCA-based optimization model that comprises over 400 Life Cycle Inventory (LCI)-compliant datasets of best available technologies for large-volume chemicals and plastics . The optimization model seeks to find the lowest-carbon avenue based on the available technologies and generates technically feasible energy and material flows throughout the chemical supply chains. These energy and material flows are based on different processes available to produce the desired chemicals. In this publication, the model is used to represent the expected production of 20 large-volume chemicals by 2030. These large-volume chemicals include ammonia, aliphatic hydrocarbons , aromatic hydrocarbons , oxygenates , polyolefins  as well as caprolactam, acrylonitrile and vinyl chloride Table . In fact2-equivalent (2.3 Gt CO2-eq.) per annum by 2030  plus the partial oxidation of hydrocarbons to oxygenates (30%). These processes should therefore receive directed efforts from the scientific community to develop alternative production processes with reduced carbon footprint. The remainder arise from the production of polyolefins (28%), aromatics (12%), chlorine (3%), vinyl chloride (3%), and acrylonitrile (2%).The model shows that the total cradle-to-gate GHG emissions of the global chemical industry will reach 2.3 billion tons of CO030 Fig. . Over ha2 feedstock for NH3 manufacture, over-oxidation of hydrocarbons to CO2, and fossil-based heat and electricity supply required to reach thermocatalytic process conditions  to NH3 via the Haber-Bosch reaction. This process accounts for 0.4 Gt CO2-eq. or 18% of the global chemical industry emissions  replacing steam-methane reforming is 0.26 t CO2-eq. per MWh of electricity , (2) using electricity only from renewable sources, without further changes to the manufacturing processes, and (3) funneling all H2 production to renewable-energy-powered water electrolysis. For this purpose, we use the same LCA model as in the previous section but limit the scope to the final demand for NH3 and oxygenates for the year 2030 . In Scenario 3, hydrogen is produced by water electrolysis. In Scenario 4, coupled electrolyzer systems are added to produce oxygenates . As the global demand of hydrogen for NH3 manufacture exceeds the demand of oxygenates, the remaining hydrogen demand unmet by the coupled electrolyzer systems is fulfilled by water electrolysis in Scenario 4. To first establish the maximum potential for GHG reductions, the renewable energy is assumed to be of zero-carbon intensity and the electrochemical routes of ideal efficiencies. With fossil-based Scenario 1 as the benchmark, Scenario 2 can reduce GHG emissions by 11%, while Scenario 3 can enable further reductions up to 53% by using hydrogen , which is not available in most industrialized countries today or even in 2030. For Scenario 4, the GHG emissions decline non-linearly; below 200 g CO2-eq. per kWh, the supply chain of NH3 manufacture switches fully from fossil- to renewable-electricity-based hydrogen production. Thus, the supply chain in Scenario 4 is based on the combination of hydrocarbon-to-oxygenate reactions and water electrolysis to produce hydrogen. More importantly, the optimization approach further determines that coupled electrolyzer technologies have the potential to reduce GHG emissions irrespective of the carbon intensity of electricity supply. GHG emissions can be reduced by up to 39% even with today’s electricity supply in the United States or China  are produced jointly and thus offer excellent conditions for the implementation of the coupled electrolyzer technologies.An analysis of state-of-art databases shows that 18% of global chemical sites currently conduct both NH17, amines to nitriles19, and tetrahydroisoquinolines (THIQs) to dihydroisoquinolines (DHIQs)20, which are thermodynamically more favorable than OER . Taking the anodic production of ethylene oxide and propylene oxide for example, technoeconomic analysis showed that high current densities  are required to minimize the surface area of electrochemical reactors and thereby lower capital costs27.Target products have been achieved with high Faradaic efficiencies (>90%) Fig. ; however50% Fig. . High st2 per MWh of renewable electricity (Table 2 per kWh). At 25% energy efficiency, the GHG reduction efficiency is 0.33 t CO2 per MWh of renewable electricity, still >25% higher than for water electrolysis at 0.26 t CO2 per MWh.High energy efficiencies indicate the yield of desired chemical products for a given electrical input, particularly that of limited renewable electricity. Energy efficiency is defined as the ratio between the useful output of the electrolyzer system and the electricity input, and is related to parameters such as conversion, Faradaic efficiency, and the voltage required to drive a given current density . To quantify the energy efficiencies required to meet the carbon reduction target, we perform a sensitivity analysis based on Scenario 4 with electricity of different carbon intensities Fig. . As mentty Table . AlthougLastly, we note that the anodic partial oxidation of higher carbon reactants, such as the oxidation of benzene to phenol and xylene to terephthalic acid, are also important towards mitigating GHG emissions from the chemicals industry. These reactions are less explored and should receive dedicated efforts from the scientific community.II salt with PtIV as a stoichiometric oxidant, in an electrochemical setting in which the anode acts as the oxidant23.To accelerate the realization of the coupled electrolyzer technology from Scenario 4, we highlight technological targets that must be achieved by researchers who wish to embark on this burgeoning research direction. An important technological target is the activation of saturated C-H bonds without the need for thermal input, in order to enable the functionalization of hydrocarbons such as methane. A potential solution is the adaptation of the Shilov system, in which the partial oxidation of a hydrocarbon such as methane to methanol or methyl chloride is catalyzed by an aqueous Pt24. The dihydroxylation of unsaturated C=C bonds appears to be favored over an activated palladium anode under neutral conditions and operating at relatively higher potential of 1.7 V vs RHE, with 81% Faradaic efficiency at 7.1 mA/cm2 towards ethylene glycol and 78% at 5.6 mA/cm2 towards propylene glycol. The activation of the palladium, to an oxidized form during the first two hours, increases the current density by ~3 fold and Faradaic efficiency by ~8 fold.In the case of longer chain hydrocarbons, it can be challenging to selectively oxidize the desired C atom. In the case of propylene, two potential sites for partial oxidation exist Fig. ; the all2. Control of operating potentials and electrolyte conditions is used to suppress OER and funnel Faradaic efficiency towards the desired product 39, followed by base-catalyzed dehydration and Cannizzarro rearrangement to form lactic acid40. The aforementioned examples show that, even with the maturation of CO2 utilization and biomass-processing technologies, anodic oxidations will be a central element of a fully renewable chemical industry.Anodic upgrading can also be used to convert biomass-derived small molecules to plastic precursors and enable sustainable polymers. The sugar derivative hydroxymethylfurfural (HMF) can be anodically oxidized to 2,5-furandicarboxylic acid (FDCA), a potential replacement for terephthalic acid in polyethylene terephthalates (PET)41 and a recent publication by the authors8. The model can be used to calculate the environmental impact of large production systems and complete supply chains for chemicals and plastics while being in-line with the ISO standards for LCA43. The TCM relies on the general calculation method of LCA44 and adapts this calculation method to represent a mathematical optimization problem, as shown in Eqs. The bottom-up model is based on the methodology of the Technology Choice Model (TCM)entclass1pt{minima45. Thus, the objective function represents the accumulated equivalent CO2 emissions. Equation 46 whenever there are no additional licenses required. There are 429 variables (processes in A) and 174 constraints (flows in y) included in the analysis. c is set to infinite.The elements in A and elementary flow matrix B from process data on the level of individual processes. These processes are either unit or aggregated datasets.For the model, we construct the technology matrix Unit process datasets are the smallest possible entity of a process with inputs and outputs. On this unit level, the dataset considers all technical inputs and outputs. Only the elementary flows of a single unit process are represented by the dataset.Unlike unit process datasets that represent only a single unit process, fully terminated aggregated process datasets represent the entire production chain. For aggregated process datasets, elementary flows enter or exit, and only one technical flow exits the process. Other technical flows are created or consumed within the production chain.41. In this procedure, commercially available technologies that lead to the lowest greenhouse gas emissions, which are denoted best available technologies48, are first identified. The mass and energy balances for all best available technologies are then included as unit process datasets. Next, we identify relevant renewable ammonia production technologies, as well as coupled anodic hydrocarbon-to-oxygenates and cathodic hydrogen evolution reactions based on literature review. We then include the unit processes for these technologies (see the following sections for further details). Lastly, aggregated datasets are included for all missing inputs  not provided by one of the unit processes already included in the previous steps.The methodology proposed by Kätelhön et al. is used to select the respective chemical and plastic production technologies8.A list of datasets can be found in Table 15 and represent the global production mix . If a global dataset is not available, a European dataset is used instead.Aggregated datasets are from the LCI database ecoinvent49. This database contains process simulations and datasets that have been verified by industrial experts. As it does not include elementary and waste flow, such as inorganic or organic residues, we adapted and included a model for waste incineration51, based on data on hazardous waste incineration in a chemical park in western Germany. In this model, flue gas cleaning is based on wet scrubbers, electrostatic precipitators, and SCR-low dust and SNCR DeNOx stages.Datasets for conventional production routes are obtained from the IHS Process Economics Program3 is produced through the Haber-Bosch process, which uses a gaseous mixture of H2 and N2 as the feed to a reactor operating at 450 °C and 200 bar. After the reaction, liquid NH3 is cryogenically separated from the product mixture. Unconverted H2 and N2 are recycled back to the Haber-Bosch reactor52. Our model utilizes the unit process data from an existing scientific publication53. The H2 feed is either supplied by water electrolysis or other H2 production processes, while N2 is supplied by an air separation unit.NH2 and O2 production, we use the process data of low-temperature water electrolysis. The average efficiency of this process is 67.15% based on net calorific values, resulting in an electricity consumption of 178.7 MJ per kg of H2 produced7.To model electrochemical HIn this publication, electrolyzer systems that couple cathodic HER with anodic hydrocarbon-to-oxygenate conversion have been added to the existing model of the chemical industry. As coupled electrolyzer technologies are still in the early development stages, unit process data for these reactions is therefore unavailable. To fill these data gaps, we assume stoichiometric reactions with full conversion for the unit processes:The electricity requirement The final demand is the total output or the amount of waste treatment of an intermediate flow. In this publication, two final demands are considered:(1) the final demand for the entire chemical industry, which is used to derive the results shown in Fig. (2) the final demand for ammonia and oxygenates only, as given in Table The GHG reduction efficiency of scenario 54. Figure This scenario-specific definition of the GHG reduction efficiency generalizes the technology-specific definition by Sternberg et al., in which the GHG emissions of a new technology is compared to that of an incumbent technologySupplementary Information