Chat), we report that local elimination of acetylcholine (ACh) in medial habenula (MHb) neurons alters glutamate corelease and presynaptic facilitation. Electron microscopy and immuno-isolation analyses revealed colocalization of ACh and glutamate vesicular transporters in synaptic vesicles (SVs) in the central IPN. Glutamate reuptake in SVs prepared from the IPN was increased by ACh, indicating vesicular synergy. Mice lacking CHAT in habenular neurons were insensitive to nicotine-conditioned reward and withdrawal. These data demonstrate that ACh controls the quantal size and release frequency of glutamate at habenular synapses, and suggest that the synergistic functions of ACh and glutamate may be generally important for modulation of cholinergic circuit function and behavior.A great deal of interest has been focused recently on the habenula and its critical role in aversion, negative-reward and drug dependence. Using a conditional mouse model of the ACh-synthesizing enzyme choline acetyltransferase ; this creates a neural circuit that is important for the brain’s responses to nicotine.The habenular-IPN circuit is rich in receptors for a neurotransmitter called acetylcholine. These receptors are also activated by nicotine, the addictive component of tobacco. Neurotransmitters are chemicals that transmit a signal from one nerve to another. These chemicals are packaged into small structures called vesicles, which are found at nerve endings. When a nerve impulse reaches the end of a nerve, it triggers the release of a vesicle’s contents into the gap (or 'synapse') between nerve cells. The released neurotransmitters can then bind to receptors on the neighboring nerve cells before being cleared away.The nicotinic acetylcholine receptors in the habenula-IPN circuit are associated with nicotine dependence in mice and humans. Frahm, Antolin-Fontes, Görlich et al. have now investigated what happens when the gene encoding the enzyme that makes acetylcholine is removed from habenular nerves in mice. The experiments revealed that these mutant mice become insensitive to the rewarding properties of nicotine and are protected from the effects of its withdrawal, following long-term exposure. The loss of acetylcholine from habenular nerves was also found to reduce the generation of small electrical currents in the nerves of the IPN. These currents are generated by another neurotransmitter called glutamate. If brain slices from normal mice are infused with acetylcholine or nicotine, these currents become more frequent. This response is not seen in brains of the mutant mice suggesting that acetylcholine helps the release of glutamate from habenular nerve endings.Frahm, Antolin-Fontes, Görlich et al. then found that the proteins that transport glutamate and acetylcholine into synaptic vesicles are found at the same sites in nerve endings in the IPN. Further experiments showed that acetylcholine also increases the reuptake of glutamate into synaptic vesicles and controls the amount and frequency of glutamate released at habenular synapses.These results thus reveal how acetylcholine mediates the effects of nicotine on the brain, in part by regulating the uptake and release of glutamate by habenular nerve endings, identifying a new mechanism important for nicotine dependence. Since many acetylcholine-releasing nerve cells also release glutamate, a future challenge will be to investigate whether the interaction between these two neurotransmitters is important for other processes that rely on acetylcholine, such as memory and thought.DOI:http://dx.doi.org/10.7554/eLife.11396.002 Acetylcholine (ACh) was first described in the heart muscle , and latAlthough a clear role for ACh in volume transmission and for nAChRs in nicotine dependence has been established, several observations suggest that ACh may have additional functions in cholinergic neurons. For example, it has been demonstrated in several systems that neurotransmitters can be coreleased , and thaChat) in MHb neurons in conditional knockout (cKO) mice. Immunogold electron microscopy revealed that ACh and glutamate vesicular transporters colocalize in a significant fraction of SVs in the central IPN (IPC). Patch clamp recordings showed that glutamatergic miniature excitatory postsynaptic currents (mEPSCs) were smaller in IPC neurons of ChAT-cKO, but unchanged in neurons of the lateral IPN (IPL) which receives non-cholinergic input, indicating that glutamate release is reduced in the absence of ACh. Upon ACh or nicotine superfusion, wild-type (wt) IPC neurons displayed an increased frequency of mEPSC. In ChAT-cKO slices this response was not observed, indicating that presynaptic facilitation of glutamate release is impaired. Direct measurements of glutamate reuptake into IPN SVs in the presence and absence of ACh demonstrated vesicular synergy. Behaviorally, ChAT-cKO mice were insensitive to the rewarding properties of nicotine and displayed no withdrawal signs after cessation of chronic nicotine treatment. Our results, therefore, establish an essential role for ACh corelease with glutamate in habenular neuron function and reveal an additional mechanism that may play an important role in nicotine dependence.To investigate the interactions between glutamate and ACh in the MHb-IPN circuit, we locally eliminated ACh at this synapse by genetic deletion of the ACh-synthesizing enzyme choline acetyltransferase  to β-actin-Cre transgenic mice die at birth CosGt(ROSA)26Sor mice (Kiaa1107-Cre mice crossed to reporter tm1(EYFP)CosGt(ROSA)26Sor mice , laterodorsal tegmental nucleus (LDTg), third cranial nerve (3N) and nucleus of the solitary tract (NTS) . Null muat birth . Therefo profile  and seleCos mice  to verifCos mice . Double Cos mice  demonstrCos mice . These rChat gene (flox/floxChat), where exons 3 and 4 are flanked by loxP sites, generates a Chat-/- allele when these exons are excised by Cre-recombinase , raphe nuclei, locus coeruleus, periaqueductal grey (PAG) and nucleus of the diagonal band. Studies on the neurotransmitter content of these other inputs indicate that there are only three sources that send cholinergic and glutamatergic projections to the IPN: the MHb, the LDTg and the nucleus of the diagonal band. Importantly, in both the LDTg and the nucleus of the diagonal band, none of the glutamatergic cells overlap with cholinergic markers . This imWe next wanted to evaluate whether the vesicular transporters for ACh and glutamate, VACHT and VGLUT1, which colocalize in the IPC by light microscopy , actuallTo determine whether they were differentially distributed across pools of synaptic vesicles, we measured the distance between each VACHT and VGLUT1-labeled vesicle and the center of the closest active zone . There wFinally, to determine whether VGLUT1 and VACHT are present in the same synapses and synaptic vesicles, we performed double post-embedding immunogold electron microscopic analyses . We obseGiven our finding that glutamate and ACh can be cotransported into SVs , and thaChat in the MHb would not directly affect expression of any cholinergic genes in the stria medullaris, which gives rise to the presynaptic cholinergic input to the MHb (We first measured evoked excitatory postsynaptic currents (eEPSCs) by local puff application of nicotine . In MHb neurons, nicotine elicits large eEPSCs (>2000 pA) that are blocked by nAChR blockers  and are  the MHb . We next the MHb –6, respo the MHb , and tha the MHb , demonstA-type receptors (A receptor blockers and tetrodotoxin (TTX)  . TTX bloin (TTX) . mEPSC win (TTX)  indicatiin (TTX) . This coin (TTX) . Quantitin (TTX) . The facIt has been reported that presynaptic nAChRs facilitate glutamate release in a variety of synapses, including MHb-IPN terminals . To testTo confirm that glutamatergic inputs to the IPN that are non-cholinergic are not affected by selective elimination of ACh in habenular neurons, we performed electrophysiological recordings in the IPL , which rTo determine if indeed ACh promotes the uptake of glutamate into SVs we assessed the levels of vesicular transporters in ChAT-cKO and wt mice by WB and coimmunoisolations, and performed glutamate uptake experiments. Western blot analyses of isolated SVs revealed no differences in the protein levels of Synaptophysin (Syp), VACHT, VGLUT1, and VGLUT2 between wt and ChAT-cKO mice  indicati+) phenylhydrazone; inhibitor of vesicular transporters). Under these conditions, the accumulation of [3H]-glutamate in IPN vesicles increased and reached a plateau of 38 ± 3 pmol/ mg of protein at 10 min . The increase by NH4+ was much more pronounced than with ACh possibly because NH4+ addresses all glutamatergic SVs present in the IPN preparation, while ACh can only increase glutamate uptake in those SVs equipped with VACHT and VGLUT. Altogether, our results show that ACh synergizes vesicular filling of glutamate, providing additional functional evidence for the coexistence of VACHT and glutamate vesicular transporters in a subset of IPN vesicles.We next analyzed glutamate uptake into SVs isolated from rat IPN. Given that VACHT and VGLUTs are coexpressed in cholinergic SVs  and that+) . During +)  (Hnasko +) . The addcting Δψ , indicatcting Δψ , the rect 10 min . In the  protein . Thus, aGiven that local elimination of ACh in habenular neurons reduces glutamate corelease  and impaTolerance to nicotine and withdrawal severity are both critical for the development and maintenance of dependence in humans and animal models . NicotinTo further study the motivational properties of nicotine, we performed the conditioned place preference (CPP) test at different nicotine concentrations. Robust preference for the nicotine-paired environment was seen in wt mice after three conditioning sessions with 1.5 mg/kg nicotine compared to the saline-paired environment . In agreOur brain is controlled by precisely regulated quantities of neurotransmitters and hormones that shape plasticity and control responses to external and internal stimuli. Alterations in the balance of neurotransmitters influence our capacity to cope with addiction, depression and, in the most pronounced cases, can contribute to psychiatric disorders. The studies presented here reveal that ACh production at MHb-IPN synapses is essential for the establishment of nicotine dependence, and that local ACh action plays an important role in withdrawal responses. We demonstrate that loss of ACh selectively in habenular neurons results in a reduction in the content of glutamate in synaptic vesicles isolated from the IPN, supporting the recent finding that MHb neurons corelease ACh and glutamate . We provTorpedo californica (The cotransmission of two neurotransmitters is not uncommon in the nervous system (for reviews see ). There ifornica . CotransOur studies agree with previous work supporting the coexistence of two modes of transmission: wired and volumetric for glutamate and ACh respectively. In particular, it has been shown that brief stimulation of MHb efferents elicits glutamatergic but not cholinergic responses, and that tetanic stimulation is required to generate slow inward currents mediated by nAChRs . These dSeveral nAChR subtypes in the MHb-IPN pathway are key in the control of nicotine consumption. Receptors containing the β4 and/or α5 subunit contribute to the activation of this tract during nicotine intake and are critical for the aversive (reward-inhibiting) effects of high-dose nicotine , while αPresynaptic nAChRs at habenular synapses facilitate glutamate and ACh release , and posCHRNB3-CHRNA6 and CHRNB4-CHRNA3-CHRNA5 clusters), and in nicotine-metabolizing enzymes (CYP2A6 and CYP2B6)  . In addiessation . The stuessation . After ressation . Similaressation  and subsflox/floxChat mice (Kiaa1107-Cre mice (founder KJ227 GENSAT) CosGt(ROSA)26Sor  reporter line was crossed to Kiaa1107-Cre line. Adult rats  were used for synaptic vesicle isolation experiments. Mice were housed with ad libitum access to food and water in room air conditioned at 22–23°C with a standard 12 hr light/dark cycle, maximal five animals per cage. All procedures were in accordance with ethical guidelines laid down by the local governing body.lox mice  were cro GENSAT) . Kiaa110(-) Nicotine hydrogen tartrate salt and mecamylamine hydrochloride were purchased from Sigma-Aldrich . Drugs were dissolved in 0.9% saline and administered intraperitoneally (i.p.) in a volume of 100 µl per 10 g body weight. Nicotine concentrations refer to the free base.Immunohistochemistry was performed as described by  in adultKiaa1107-Cre crossed to tm1(EYFP)CosGt(ROSA)26Sor mice. 8 to 16 sections from three different mice were used for analysis. The number of CHAT positive neurons per section was quantified in wt and ChAT-cKO mice. 13 to 22 sections from three different mice per genotype were used for analysis.Image J was used for quantification analysis. The number of CHAT and EYFP cells per section was quantified in Images used for colocalization analysis were acquired with a 40× oil immersion objective on a Zeiss LSM700 confocal microscope. The analysis was done as described in . Briefly2 and 5% CO2) artificial cerebrospinal fluid containing (mM): 125 NaCl, 2.5 KCl, 1.3 MgCl2, 2 CaCl2, 1.25 KH2PO4, 11 glucose and 26 NaHCO3 . The internal pipette solution contained (mM): 120 potassium gluconate, 2 MgCl2, 0.5 CaCl2, 5 EGTA and 10 Hepes . Nicotine was locally applied by pressure  with pipettes similar to the recording pipette using a pressure regulator  controlled with a trigger interface . The pipette was moved within 15–20 μm of the recorded cell using a motorized micromanipulator  for drug application and retracted at the end of the puff to minimize desensitization.For recordings of evoked excitatory postsynaptic currents (eEPSCs) from MHb and IPN somata, slices were cut at 350 µm from adult mice, on a Microm HM 650 V (Thermo Scientific). Slices were kept submerged at room temperature in oxygenated , preincubated for 30 min at 37°C and then transferred to the recording solution containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1.3 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 10 glucose, 2 sodium pyruvate, 3 myo-inositol, 0.44 ascorbic acid, bubbled with a mixture of 95% O2/5% CO2. Slices rested in this recording solution at room temperature for at least one hour before they were transferred to the recording chamber and perfused with ACSF containing 1 µM TTX, 100 µM Picrotoxin, and 1 µM CGP 55,845 hydrochloride . Electrophysiological responses were recorded with an EPC 10 patch-clamp amplifier and PatchMaster and FitMaster software (HEKA Elektronik). Cells were patch-clamped at -70 mV for 10 min, before 100 µM ACh or 250 nM nicotine tartrate (Sigma Aldrich) was washed in for 5 min. Spontaneous mEPSCs were analyzed for the one minute before ACh or nicotine application (baseline), and for the last minute of drug application with Mini Analysis . Patch pipettes had resistances of 4–8 MΩ when filled with a cesium-based solution containing (in mM): 105 CsMeSO, 30 CsCl, 10 Hepes, 10 phosphocreatine, 2 ATP-Mg2+, 0.3 GTP (pH adjusted to 7.2 with CsOH). In recordings of the IPL, 3–5 mg/ml Biocytin (Sigma Aldrich) was included in the intracellular recording solution. After recordings, slices were fixed with 4% PFA for 30–45 min, and incubated with a streptavidin conjugated Alexa Fluor 488 dye  to label biocytin filled neurons, and a rat primary antibody for substance P  to label the IPL. After incubation with secondary antibodies, fluorescent signals were detected using a confocal laser-scanning microscope (Zeiss). Results are presented as means ± SEM.For electrophysiological recordings of mEPSCs in the IPN, adult mice were sacrificed by cervical dislocation and brains were dissected in chilled (4°C) artificial cerebrospinal fluid (ASCF) containing (in mM): 87 NaCl, 2 KCl, 0.5 CaClThe MHb and IPN were dissected from adult ChAT-cKO and wt mice (n = 3 per genotype), collected in 1 ml of lysis buffer  and immediately homogenized. The homogenates were processed as described in . PrimaryAdult mice were perfused transcardially with 0.9% NaCl for 1 min followed by fixative containing 4% PFA in 0.1M sodium cacodylate buffer (pH 7.4) for 10 min. The brains were kept in the fixative overnight at 4°C and sectioned coronally with a vibrating microtome  at 50 µm thickness the following day.en bloc staining with 1% uranyl acetate for 30 min, dehydration in a graded series of ethanol, infiltration with Eponate 12™ Embedding Kit , embedding with the resin and polymerization for 48 hr at 60°C. Series of 90 nm ultrathin sections were cut and analyzed on a JEOL JEM-100CX at 80 kV using with the digital imaging system .IPN coronal sections were incubated in blocking solution  in the buffer for 2 hr at room temperature. The sections were then single labeled at 4°C overnight with the following primary antibodies diluted in blocking solution: rabbit polyclonal serum against the vesicular acetylcholine transporter   and guinIsolated IPN from 50 µm vibratome sections were applied to a high pressure freezer  in cryoprotectant (30% BSA) and transferred to a freeze substitution unit . They were incubated with a substitution medium, containing 1% uranyl acetate, 95% acetone and 5% water, at -85°C, washed in acetone, infiltrated with Lowicryl HM20 and embedded in the resin at -45°C. Ultrathin sections (90 nm) were cut and processed for post-embedding immunolabeling.Post-embedding immunolabeling was carried out on 90 nm ultrathin sections mounted on carbon film 200 mesh nickel grids . Sections were incubated in blocking solution containing 3% BSA, 0.1% saponin in 20 mM Tris (pH 7.5) for 2 hr at room temperature, incubated with single or double primary antibodies   and/or gQuantification was performed on preembedding nanogold labeled synaptic terminals (at 20000 magnification) only when active zone, synaptic cleft and vesicles were clearly presentable . In a separate analysis, the distance between a VGLUT1/VACHT-gold particle and the nearest VGLUT1/VACHT-gold particle was measured using electron micrographs with postembedding double immuno gold labeling . Both measurements were done with ImageJ.2O, in mM:10 HEPES/KOH/pH 7.4, 1 PMSF, Pi 1:1000) for osmotic shock and processed with three strokes at 2000 rpm using a Wheaton homogenizer to obtain SVs in suspension. Immunoisolation of SV was performed using paramagnetic beads  were first coated with the respective primary antibodies suspended in coating buffer , supplemented with 0.5 – 1 µg of IgG per 107 Pan Mouse IgG beads or 2 x 107 M-280 sheep anti-rabbit beads and rotated for 2 hr at 4°C followed by four washing steps. Immunoisolation was performed overnight at 4°C under rotation using a suspension of the coated beads and S1shock fractions diluted with incubation buffer  to yield a ratio of 50–75 µg protein to 1.4 x107 beads (Pan Mouse IgG) or 3.9 x 107 beads (M-280). SV bound to beads were washed three times in incubation buffer, and three times in coating buffer. Bead-bound SV were finally dissolved in sample buffer (Ten mice per genotype (ChAT-cKO and wt littermates) were used to dissect IPN brain samples. Brain tissue was resuspended in ice-cold buffer  1:1000 (Sigma-Aldrich) and homogeneized ). Nuclei were discarded by centrifugation and the supernatant mainly containing SVs was diluted 1:10 in lysis buffer (ddHe buffer . Beads w4 (corresponding to 1 mM free Mg2+), 2 Na-ATP, adjusted to pH 7.0 with KOH) by adding 1 μM neostigmine, 49.5 µM K-glutamate and 0.5 µM -L-glutamic acid  to the SV suspension. Non-specific uptake was performed by adding 60 µM FCCP also used for background correction. Additives were given 10 min before the uptake was started at 25°C. The reaction was stopped after 10 min by adding ice-cold buffer containing the same ionic constituents used during the uptake. Non-bound radioactivity was removed by centrifugation at 435,000 x g  for 10 min, and the pellets were washed three times. Radioactivity was measured by liquid scintillation counting .  from 10 rat IPN. Briefly, membranes of S1shock fraction (see above) were removed  and SV were pelleted ) resulting in pLP2 fraction. Uptake was performed in K-gluconate buffer . .The MoTil system  was used to monitor open-field behavior of 2–3 month old male mice in complete darkness (20 x 40 x 40 cm black box). Single events represented disruption of two distinct infrared photobeams 3 cm apart in the cage. After injection of saline or different concentrations of nicotine, the number of beam breaks was measured for 15 min. Locomotor inhibition was calculated by normalizing to basal activity values . For measurement of tolerance behavior, mice were given daily i.p. injections of 0.65 mg/kg nicotine , starting with saline at day 0 as control activity. Activity was monitored in the open field box every second day for 15 min immediately after injections.Home cage activity of single housed animals was measured for 3 days and averaged to 24 hr.A rectangular three-compartment box separated by removable doors (TSE-systems) was used. The center compartment (6 x 15 x 20 cm) is grey with a polycarbonate smooth floor. The choice compartments (17 x 15 x 20 cm) have different visual and tactile cues. Time spent in each compartment was measured with photobeam detectors. During the preconditioning phase (day1) male mice (8–12 weeks old) were placed into the center compartment with closed doors. After 2 min of habituation, doors opened automatically and mice were allowed to explore the three compartments freely for 15 min. The pre-conditioning session was used to determine baseline responses and less preferred compartments were paired with nicotine. Mice exhibiting a strong preference for one side (>150 s) in the pre-conditioning session were excluded. During the conditioning phase (day 2–4), mice were given a saline injection in the morning and a nicotine injection in the afternoon and were confined to either one side or the other of the conditioning box for 20 min. On the preference test (day 5), the doors between the compartments were opened after mice habituated for 2 min in the central chamber and were then allowed to move freely in the three compartments for 15 min. Preference score was measured as time spent in nicotine-paired or saline-paired compartment at day 5 – day 1.Single housed male adult mice were treated with nicotine via the drinking water over 6 weeks, starting at a concentration of 32,5 µg/ml in the first week, increasing to 65 µg/ml in the second week and to 162,5 µg/ml in the remaining 4 weeks . To miniStatistical analysis was performed with GraphPad Prism 6.0. Unpaired two-tailed Student t-tests were used for analyzing most of the data, except when two-way analysis of variance with ANOVA or paired two-tailed Student t-tests are indicated. Results are presented as means ± S.E.M. review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and two reviewers, one of whom, Sacha Nelson, is a member of our Board of Reviewing Editors. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Thank you for resubmitting your work entitled "An Essential Role of Acetylcholine-Glutamate Synergy at Habenular Synapses in Nicotine Dependence" for further consideration at 1) The assumption that two gold particles within a distance of 90 nm label the same vesicle is likely to give an overestimate of the number of vesicles containing both VAChT and VGLUT1, because gold particles on adjacent vesicles can easily be less than 90 nm apart given the close packing. The estimate of double-positive vesicles may be an overestimate, making it difficult to accept the authors' claims that the majority of SVs in these axon terminals contain both transporters. The authors should present a more compelling argument for why 90 nm is not only the maximum distance for labeling in the same vesicle but also the minimum distance for labeling in adjacent vesicles, or should make textual changes acknowledging the colocalization may be lower.2) Given the similarity of the numbers at the bottom of the subsection “Cholinergic and glutamatergic inputs to IPN subnuclei” it would be appropriate to add the word "slightly", so that the sentence reads "slightly" less overlap in the IPI.3) The authors' claim that ChAT deletion resulted in the "complete absence" of nicotine-elicited behavioral responses is too strong of an assertion. Nicotine was still able to elicit hypolocomotion in the ChAT-cKO mice, albeit to a lesser degree than WT mice . The auteLife. Your full submission has been evaluated by a Senior editor and three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you the manuscript in its present form will not be considered further for publication in eLife.Thank you for choosing to send your work entitled "An Essential Role of Acetylcholine-Glutamate Synergy at Habenular Synapses in Nicotine Dependence" for consideration at eLife to reject manuscripts for which the additional work required would be likely to take several months or longer. However, in this case, we would encourage you to submit a new manuscript that addresses the concerns raised by the authors. We will endeavor to send the manuscript back to the same reviewers, although there is no guarantee that this will be possible.As you will see all three reviewers were enthusiastic about the work but felt that additional experiments were needed to support the core conclusions of the paper. Since some of these involve breeding additional animals we recognize that it is unlikely that this can be completed in a brief period (i.e. two months). It is the policy of Although you should address all of the points raised, the reviewers and editors agreed that the following experimental issues were the most essential:1) The driver strain should be better characterized. At a minimum you should cross the Cre line to a reporter mouse and perform a Chat stain. This would allow you to then quantify both the penetrance and specificity of the line.2) You need to at least make a better argument for why non-habenular glutamatergic inputs are unlikely and may need to do some additional experiments to shore up this point.3) You need to better quantify and better support the western blot data, perhaps adding measures of holding current and paired pulse responses.4) Additional controls are needed for co-localization experiments. This can probably be done simply by reanalyzing the images already acquired.5) The sample size for Reviewer #1:In this paper the authors use conditional deletion of CHAT to convincingly demonstrate synergy between ACh and glutamate at MHb synapses as well as showing changes in behavioral correlates of tolerance and withdrawal. These are important and interesting results.1) My major concern with the paper is that the authors have not demonstrated that this synergy is the only, or even a necessary component of mechanism of the behavioral changes they observe. Given the higher affinity of NMDA receptors than many other glutamate receptors, it is not clear that they would not be activated in the absence of facilitation of glutamate release.The issue may be somewhat semantic – that is the authors may wish "synergy" in the title and Discussion to apply more broadly to any sort of interaction between ACh and glutamate, as opposed to the vesicular synergy on which they focus for much of the results.2) A western blot  is not aReviewer #2:The study described in Frahm et al. investigates the mechanism and functions of glutamate/acetylcholine co-release from MHb inputs onto interpeduncular nucleus (IPN) neurons. The authors have an excellent model for the analysis of MHb neurons as the cre line they describe may be specific for cholinergic neurons in the MHb. They go on to show that deletion of Chat from Cre+ neurons alters mEPSC amplitude in the IPN, and that VAChT and vGlut1 are both expressed on synaptic vesicles in terminals of the IPN. Finally, they show that this mouse line has an attenuated hypolocomotor response to nicotine injection and a lack of nicotine preference in a place preference task. While the authors offer some intriguing results there are major flaws in some of their conclusions. The study would also greatly benefit from a more thorough and quantitative characterization of their MHb/Chat cKO mouse model.1) While the A830010M20Rik-Cre line may only express Cre in cholinergic neurons of the MHb, it clearly expresses Cre in many other areas of the brain. Quantification of the number of Chat+ neurons in the WT and Chat-cKO in the MHb is required at a minimum.2) If nicotinic responses are still observable in IPN neurons when nicotine is directly applied , then th3) While the authors show that mEPSC amplitude is decreased in IPN neurons of the Chat-cKO, mEPSC recordings sample from all input that a neuron receives, not just the inputs from MHb. The authors need to show that glutamatergic currents from MHb are affected in the Chat-cKO.4) In 5) The authors state that the quantity of vesicular proteins in WT and cKO are similar in 6) The physiology experiments in 7) I am fundamentally confused about the logic of analyzing nicotine-induced responses in an animal that can't release Ach. Why would one look for or expect changes in a post-synaptic dependent mechanism given a pre-synaptic manipulation? Or is the logic that the nicotine induced behaviors all act via presynaptic receptors that regulate glutamate release?Reviewer #3:The paper does a good job of going from biophysics to behavior. As in all such studies, there are weaknesses; but on the whole the story is good."This was expected, since cholinergic input from the stria medularis to the MHb  is not affected by conditional deletion of ChAT in the MHb" should be, "This was expected, since conditional deletion of ChAT in the MHb would not directly affect expression of any cholinergic genes in the stria medullaris, which gives rise to the presynaptic cholinergic input to the neurons studied. .”The paragraph beginning "we next analyzed glutamatergic re-uptake" has some errors. More important is the conclusion that ChAT KO changed vesicular contents-the conclusion reached by comparing the change in mEPSC amplitudes vs. no change in GluR1 westerns. The conclusion is weak here, but subsequent data strengthen it. The westerns deal with both synaptic and extrasynaptic GluR1, of which a small number might be at the synapses of interest. Did perfusion with Ach or nicotine induce changes in holding currents?More importantly, the analysis of colocalization is incomplete, because it lacks the controls: the authors don't show large-large and small-small gold particle distance distributions for comparison. In The Discussion is overall sensible, but the final hypothesis may go too far. You could also consider the possibility that eliminating ChAT decreases pH in the vesicle (increases deltapH), by decreasing the number of protons leaving. This, in turn, would increase nicotine accumulation in the vesicle . What might this do?Finally, if the vesicle-loading hypothesis is correct, then the authors should phenocopy the acute electrophysiological effects with vesamicol. 1) The assumption that two gold particles within a distance of 90 nm label the same vesicle is likely to give an overestimate of the number of vesicles containing both VAChT and VGLUT1, because gold particles on adjacent vesicles can easily be less than 90 nm apart given the close packing. The estimate of double-positive vesicles may be an overestimate, making it difficult to accept the authors' claims that the majority of SVs in these axon terminals contain both transporters. The authors should present a more compelling argument for why 90 nm is not only the maximum distance for labeling in the same vesicle but also the minimum distance for labeling in adjacent vesicles, or should make textual changes acknowledging the colocalization may be lower.We have edited this part in the manuscript  to indicate that the 90 nm maximal theoretical distance between two gold particles overestimates the number of double positive SVs because it does not exclude that two particles at a distance of less than 90 nm or even juxtaposed could be labeling adjacent vesicles. We have also made changes in 2) Given the similarity of the numbers at the bottom of subsection the “Cholinergic and glutamatergic inputs to IPN subnuclei” it would be appropriate to add the word "slightly", so that the sentence reads "slightly" less overlap in the IPI.We have edited this.3) The authors' claim that ChAT deletion resulted in the "complete absence" of nicotine-elicited behavioral responses is too strong of an assertion. Nicotine was still able to elicit hypolocomotion in the ChAT-cKO mice, albeit to a lesser degree than WT mice . The autWe have edited this point in the manuscript  and the figure legend  to remov[Editors’ note: the author responses to the previous round of peer review follow.]As you had predicted, the additional experiments that were performed to address issues raised in review required several months to complete. We agree with the reviewers that additional data were required to strengthen the main conclusions of the paper, and we believe we have included all additional experiments that were requested in review. We have also extensively reorganized and revised the manuscript in response to the points raised.[…] Although you should address all of the points raised, the reviewers and editors agreed that the following experimental issues were the most essential: 1) The driver strain should be better characterized. At a minimum you should cross the Cre line to a reporter mouse and perform a Chat stain. This would allow you to then quantify both the penetrance and specificity of the line.tm1(EYFP)Cos in We have included substantial further analyses of the driver mouse line A830010M20Rik-Cre crossed to the reporter mouse line Gt(ROSA)26SorTo assess the penetrance of the driver Cre-line, we provide quantification of the number of CHAT positive neurons in ChAT-cKO mice (A830010M20RikCre x floxed ChAT) in 2) You need to at least make a better argument for why non-habenular glutamatergic inputs are unlikely and may need to do some additional experiments to shore up this point.To clarify this point we have included a detailed explanation of the inputs to the IPN (subsection “Cholinergic and glutamatergic inputs to IPN subnuclei”). This section describes that the main glutamatergic input to the IPN is from the medial habenula, but there are other glutamatergic inputs to the IPN. These include projections from the laterodorsal tegmentum (LDTg), raphe nuclei, locus coeruleus, periaqueductal grey (PAG) and nucleus of the diagonal band. The neurotransmitter content of these other inputs is as follows:a) The LDTg contains cholinergic, GABAergic and glutamatergic (VGLUT2) neurons . Importantly the vast majority of CHAT positive neurons are not glutamatergic .b) The raphe nuclei are mainly serotoninergic: only the dorsal raphe contains some glutamatergic neurons (VGLUT3) .c) The locus coeruleus produces mainly norepinephrine, however some neurons may coexpress glutamate .d) The PAG does not express VGLUT1, but does express a small amount of VGLUT2  and VGLUT3 , and is very rich in GABAergic neurons .e) The diagonal band contains three populations of neurons: cholinergic, GABAergic and glutamatergic (VGLUT2 and VGLUT3 positive) . Importantly none of the glutamatergic cells overlap with cholinergic markers .f) In addition the great majority of IPN neurons are primarily GABAergic excluding the possibility that the recorded mEPSCs are from local projections ).Our experimental arguments supporting the conclusion that non-habenular glutamatergic inputs are unlikely are the following:A) We provide new quantitation of the electron microscopy data that indicates that at least 80%  of synaptic terminals in the central part of the IPN (IPC) contain synaptic vesicles positive for both VGLUT1 and VACHT and that very few, 11%, are only glutamatergic (10/90)) new .B) As a second method to quantify the degree of overlap of glutamatergic and cholinergic terminals in different nuclei of the IPN, we calculated the Manders’ colocalization coefficient that ranges from 0 for no colocalization to 1 for complete colocalization new  (MandersC) As the published studies indicate (description of IPN inputs above), there are only three sources of cholinergic+glutamatergic projections to the IPN: the MHb, the LDTg and the nucleus of diagonal band. In both the LDTg and the nucleus of diagonal band, the neurons express either CHAT or VGLUT but not both markers in the same cell .D) Therefore in the IPC, where we have performed the EM studies and where the electrophysiological recordings show that glutamatergic mEPSCs differ in the ChAT- cKO, 80% of the inputs to the neurons we have recorded are glutamatergic+ cholinergic and thus they originate in the habenula.E) In addition, we have performed electrophysiological recordings in the lateral IPN new , which rTo clarify these points, we include new diagrams , 8 and 93) You need to better quantify and better support the western blot data, perhaps adding measures of holding current and paired pulse responses.We agree that this is an important point and have included more experimental data and edited this part in the manuscript. We agree that the information provided by the WB is not very quantitative since it does not distinguish between presynaptic, extrasynaptic and postsynaptic receptors and whether they are at the membrane or not. We have rephrased this part in the manuscript, we include additional electrophysiological measurements of holding currents and we have increased the sample size of mEPSC recordings upon nicotine and ACh bath application . These rflox/flox and Chat flox/flox must be recombined in both chromosomes . This is clearly beyond the scope of this revision, and we believe it should not be required.We have performed paired pulse responses by electrical stimulation of the fasciculus retroflexus (fr) and recordings in IPN neurons using oblique slices, which included the MHb, fr and IPN, but unfortunately there was too much variability between slices depending on the sectioning of the fasciculus retroflexus. Optogenetic recordings in ChAT-channelrhodopsin (ChR2) mice ) would have been a better alternative, because these would allow direct stimulation of the MHb terminals, versus stimulation of the fr as is needed for electric stimulations. However in our case optogenetic recordings would have required crossing 3 mouse strains (Chat-ChR2 X A830010M20Rik-Cre X Chat 4) Additional controls are needed for co-localization experiments. This can probably be done simply by reanalyzing the images already acquired.As requested, we have re-analyzed the electron microscopy data and provided distance distributions of large-large (VGLUT1-VGLUT1) and small-small (VACHT-VACHT) gold particles in co-labeling EM experiments by quantitation of the number of gold particles which are at 0-90 (same vesicle), 90-180 (next vesicle) and more than 180 nm apart .2 =16%).For VACHT-VACHT, the percentage of particles in the same vesicle is 61%. This is above random distribution (which would be 0.42 =10%).For VGLUT1-VGLUT1, the percentage of particles in the same vesicle is 36%. This is above random distribution These values indicate that the double EM labelling of small-small, large-large and small-large particles is non-random and that the majority of SVs in the central IPN contain both vesicular transporters. We have indicated this in the Results section 5) The sample size for We have increased the sample size to > 20 mice per genotype, and > 10 per condition. These data is now presented in Reviewer #1:In this paper the authors use conditional deletion of CHAT to convincingly demonstrate synergy between ACh and glutamate at MHb synapses as well as showing changes in behavioral correlates of tolerance and withdrawal. These are important and interesting results. My major concern with the paper is that the authors have not demonstrated that this synergy is the only, or even a necessary component of mechanism of the behavioral changes they observe. Given the higher affinity of NMDA receptors than many other glutamate receptors, it is not clear that they would not be activated in the absence of facilitation of glutamate release. The issue may be somewhat semantic – that is the authors may wish "synergy" in the title and Discussion to apply more broadly to any sort of interaction between ACh and glutamate, as opposed to the vesicular synergy on which they focus for much of the results.eLife audience, although we would be happy to change the title if required.The word synergy in the title is used in the broad sense described in the last sentence of this comment. We believe it is appropriately general for the A western blot  is not aThis was one of the major 5 points. We appreciate the insight from the reviewer and have included more experiments and edited this part in the Results and Discussion of the manuscript. See reply 3 of the summary points.Reviewer #2:The study described in Frahm et al. investigates the mechanism and functions of glutamate/acetylcholine co-release from mHb inputs onto interpeduncular nucleus (IPN) neurons. The authors have an excellent model for the analysis of mHb neurons as the Cre line they describe may be specific for cholinergic neurons in the mHb. They go on to show that deletion of Chat from Cre+ neurons alters mEPSC amplitude in the IPN, and that VAChT and vGlut1 are both expressed on synaptic vesicles in terminals of the IPN. Finally, they show that this mouse line has an attenuated hypolocomotor response to nicotine injection and a lack of nicotine preference in a place preference task. While the authors offer some intriguing results there are major flaws in some of their conclusions. The study would also greatly benefit from a more thorough and quantitative characterization of their mHB/Chat cKO mouse model. 1) While the A830010M20Rik-Cre line may only express Cre in cholinergic neurons of the mHb, it clearly expresses Cre in many other areas of the brain. Quantification of the number of Chat+ neurons in the WT and Chat-cKO in the mHb is required at a minimum.This was one of the major 5 points. See reply 1 of the summary points.2) If nicotinic responses are still observable in IPN neurons when nicotine is directly applied , then thThis was one of the major 5 points. See reply 3 of the summary points.3) While the authors show that mEPSC amplitude is decreased in IPN neurons of the Chat-cKO, mEPSC recordings sample from all input that a neuron receives, not just the inputs from mHb. The authors need to show that glutamatergic currents from mHB are affected in the Chat-cKO.See reply 2 of the summary points.4) In See reply 2 of the summary points. In addition we have changed “habenular terminals” for “axonal terminals” in the EM figures to reflect the fact that most presynaptic terminals but not all of them are habenular.5) The authors state that the quantity of vesicular proteins in WT and cKO are similar in We have quantified the levels of VACHT, VGLUT1 and VGLUT2 with respect to synaptophysin in wt and ChAT-cKO. These data are shown in 6) The physiology experiments in See reply 5 of the summary points indicating that the sample size was increased to > 20 mice per genotype, and > 10 per condition See also reply 3 of the summary points for the presynaptic release probability measurement.7) I am fundamentally confused about the logic of analyzing nicotine-induced responses in an animal that can't release Ach. Why would one look for or expect changes in a post-synaptic dependent mechanism given a pre-synaptic manipulation? Or is the logic that the nicotine induced behaviors all act via presynaptic receptors that regulate glutamate release?This is an interesting point. Given that nAChRs can act either presynaptically or postsynaptically, we were interested in the detailed mechanisms through which ACh acts when locally released in the CNS. Since the MHb is an extremely rich source of ACh, and since it is implicated in many different aspects of addictive behaviors, we performed this study to investigate the actions of ACh at these synapses and its effects on behaviour.Reviewer #3:The paper does a good job of going from biophysics to behavior. As in all such studies, there are weaknesses; but on the whole the story is good. We have made the suggested changes in new "This was expected, since cholinergic input from the stria medularis to the MHb  is not affected by conditional deletion of ChAT in the MHb" should be, "This was expected, since conditional deletion of ChAT in the MHb would not directly affect expression of any cholinergic genes in the stria medullaris, which gives rise to the presynaptic cholinergic input to the neurons studied. .”We have rephrased the sentence as suggested .The paragraph beginning "we next analyzed glutamatergic re-uptake" has some errors. More important is the conclusion that ChAT KO changed vesicular contents-the conclusion reached by comparing the change in mEPSC amplitudes vs. no change in GluR1 westerns. The conclusion is weak here, but subsequent data strengthen it. The westerns deal with both synaptic and extrasynaptic GluR1, of which a small number might be at the synapses of interest. Did perfusion with Ach or nicotine induce changes in holding currents?We appreciate the insight from the reviewer and have included more experiments and edited this part in the Results and Discussion of the manuscript. See reply 3 of the summary points.We have changed this.We have now separated the electron microscopy We have made the suggested changes in More importantly, the analysis of colocalization is incomplete, because it lacks the controls: the authors don't show large-large and small-small gold particle distance distributions for comparison. See reply 4 of the summary points.We have made the suggested changes and included the reference of a recent publication by two of the authors where the role of NH4+ in vesicular glutamate uptake is demonstrated .In We have changed this sentence in the manuscript .The Discussion is overall sensible, but the final hypothesis may go too far. You could also consider the possibility that eliminating ChAT decreases pH in the vesicle (increases deltapH), by decreasing the number of protons leaving. This, in turn, would increase nicotine accumulation in the vesicle . What might this do?We have changed the last part of the Discussion.Generally the transport of each negatively charged anion like glutamate or Cl activates the proton pump to insert a proton for compensation. To sustain transport even if protons are accumulating, VGLUT itself then exchanges H+ against K+ as described in our recent paper . We carefully checked the literature and there is no indication that nicotine is actively transported over the plasma membrane by the choline transporter ChT . We also found no indication that nicotine binds to VACHT or is actively taken up. So we think that even if nicotine will be distributed overall including glutamatergic terminal and vesicles, the effect seen will be on all glutamatergic vesicles/ synapses and should give an overall increase in mEPSC.Finally, if the vesicle-loading hypothesis is correct, then the authors should phenocopy the acute electrophysiological effects with vesamicol.We tried short application of vesamicol (VACHT blocker) in IPN neurons, but did not see any effects on mEPSC amplitude and/or frequency of mEPSCs in our initial experiments most likely because all the vesicles in the reserve pool have to be exocytosed before vesamicol can change the loading of the next set of SVs. Thus there is a problem with the experimental design that would require sustained activation of habenular neurons while applying TTX to measure mEPSCs. However, we think that these pharmacological experiments might not lead to further conclusions than the ones shown already with a cell specific genetic manipulation.