CB1 receptor signaling at the cingulate-striatal circuit is anxiogenic

Domain: neuroscience
Published: 2026-04-08
Editorial outcome: major_revision

Abstract
Cannabinoids, such as Δ9-tetrahydrocannabinol (THC), produce anxiety at high doses, yet the underlying neural mechanisms remain poorly understood. The anterior cingulate cortex (ACC) sends dense monosynaptic projections to the dorsomedial striatum (DMS). Optogenetic excitation of the ACC→DMS pathway promotes anxiolytic-like behavior, suggesting that its suppression could induce anxiogenic states. We hypothesized that CB1 receptor-mediated suppression of this circuit contributes to cannabinoid-induced anxiogenic effects. We found that endocannabinoid (eCB) system components are expressed in ACC→DMS neurons and that cannabinoids depress excitatory transmission in this circuit in mice. Additionally, in vivo fiber photometry revealed that anxiogenic and aversive stimuli trigger eCB release from the DMS, which activates CB1 receptors on ACC axon terminals. Notably, pathway-specific deletion of CB1 receptors in the ACC→DMS circuit reduced both innate anxiety-like behaviors and conditioned place aversion to THC in mice. Collectively, these findings identify a circuit-level mechanism whereby cannabinoid signaling promotes anxiety and aversion.

Introduction
Endocannabinoids (eCBs) are lipid-derived neurotransmitters that retrogradely depress synaptic transmission through activation of presynaptic cannabinoid receptor 1 (CB1)1, 2, 3. CB1 receptors are key regulators of affective states, influencing stress responsivity, fear learning, and fear extinction4, 5, 6, 7, 8. Pharmacological elevation of the eCB 2-arachidonoylglycerol (2-AG) by inhibiting its primary degradative enzyme, monoacylglycerol lipase (MAGL), produces anxiolytic and antidepressant-like effects in stress models9, 10, 11, 12. Conversely, global knockout of diacylglycerol lipase (DAGLα), the principal enzyme responsible for 2-AG synthesis, induced anxiety-like behavior in mice13. Together, these findings demonstrate that emotional states are bidirectionally modulated by alterations in eCB signaling.

Population studies indicate that relaxation and euphoria are the primary motivations for cannabis use14. Δ9-Tetrahydrocannabinol (THC), the principal psychoactive constituent of cannabis, acts as a non-selective partial agonist at cannabinoid receptors, with particularly high affinity for CB1, and mediates most central effects through CB1 receptor activation15, although CB2 receptors can also contribute16, 17. CB1 signaling in key regions including the amygdala, nucleus accumbens, ventral tegmental area, and bed nucleus of the stria terminalis (BNST) is well established to regulate anxiety and reward processing6, 16, 18, 19, 20. It is likely that coordinated CB1 signaling across interconnected cortical and subcortical circuits is a critical determinant of the anxiolytic and rewarding effects of low dose THC.

Paradoxically, THC produces dose-dependent anxiogenic effects in both rodents and humans. At higher doses, THC can trigger panic, paranoia, and anxiety in humans21 and induces conditioned place aversion (CPA) in rodents22. Cannabis products with high levels of THC have been linked to anxiety, which contributes to the growing proportion of cannabis-related emergency department visits as legalization has expanded23. Despite the clinical and public health relevance, the neural circuits mediating the anxiogenic and aversive effects of high-dose THC remain poorly defined.

One candidate locus is the anterior cingulate cortex (ACC), a dorsomedial prefrontal structure that integrates limbic, cognitive, and motivational signals to guide adaptive behavior under conditions of uncertainty24, 25, 26, 27, 28. In decision-making tasks, ACC activity is associated with cognitive control, error detection, and behavioral flexibility28, 29, 30. Optogenetic silencing of the ACC impairs attention28, 29, reduces error monitoring24, and promotes avoidance behavior30, which are commonly reported in cannabis users31, 32. The ACC sends dense projections to the dorsomedial striatum (DMS)33. CB1 receptors are abundantly expressed in the striatum34, 35 and are localized to glutamatergic terminals36, where their activation inhibits cortical glutamate release35, 37. Given that optogenetic stimulation of ACC terminals in the DMS produces anxiolytic-like effects30, we hypothesized that suppression of the ACC→DMS transmission through CB1 receptor activation by either THC or eCBs could promote anxiogenic or aversive states. To test this hypothesis, we employed a multi-level experimental approach. First, RNAscope fluorescent in situ hybridization was used to quantify expression of key eCB system components in the ACC and DMS. Second, using optogenetic electrophysiology, we demonstrate that 2-AG released from DMS neurons retrogradely suppresses glutamatergic input from the ACC. Third, we used in vivo fiber photometry with the GPCR-based sensor GRAB eCB2.0 to measure eCB signaling at ACC terminals in the DMS during approach and avoidance behavior. Finally, we determined how pathway-specific deletion of CB1 receptors from ACC neurons projecting to the DMS alters anxiety-like behavior and THC-induced conditioned place aversion. Together, these findings identify the ACC→DMS pathway as a critical locus at which cannabinoid signaling promotes anxiogenic affective states.

Results
The excitatory ACC→DMS projection is monosynaptic and is regulated by eCBs
We first performed brain-wide- anterograde mapping of ACC excitatory neurons by injecting AAV9-CaMKII-hChR2(E123T/T159C)-mCherry into the ACC, which revealed widespread projections to cortical and subcortical regions, including dense innervation of the DMS (Supplementary Fig. 1). To test whether these projections form functional synapses with medium spiny neurons (MSNs) in the DMS, we next carried out electrophysiological recordings in striatal slices two weeks after viral injection (Fig. 1a). Blue light stimulation (470 nm, 2 ms) of ACC axon terminals through the 40x objective evoked excitatory postsynaptic currents (EPSCs) that scaled linearly with light power (Fig. 1b) and were blocked by the AMPAR antagonist CNQX (10 µM; Fig. 1c). Optogenetic EPSCs (oEPSC) could arise from a monosynaptic connection between the ACC and DMS MSNs, or alternatively, are mediated via intermediary excitatory neurons in a polysynaptic circuit. We distinguished these possibilities by applying the Na+ channel blocker tetrodotoxin (TTX) and the K+ channel blocker 4-aminopyridine (4-AP), which block polysynaptic but not monosynaptic currents38. Bath application of TTX (1 µM) blocked opto-EPSCs, which was reversed by addition of 4-AP (1 mM; Fig. 1d). Because rescue of light-evoked currents by 4-AP occurred in the presence of TTX, the oEPSCs must be monosynaptic since no intermediary neuron would have been capable of firing an action potential. Blockade of K+ channels with 4-AP greatly enhances the ChR2-induced depolarization of axons and activation of Ca2+ channels, which are sufficient to rescue transmitter release38. Thus, the ACC makes monosynaptic connections with MSNs in the DMS.

Depolarization-induced suppression of excitation (DSE) is a form of short-term synaptic depression triggered by Ca2+ influx which leads to synthesis and release of 2-AG from postsynaptic neurons and retrograde activation of presynaptic CB1 receptors3, 39. After establishing a stable baseline of oEPSCs, a brief depolarization (from -80 to 0 mV, 5 s) elicited DSE which was blocked by the CB1 antagonist AM251 (Fig. 1e). Similarly, bath application of the CB1 receptor agonist WIN55212-2 (2 µM) depressed opto-EPSCs in DMS MSNs (Fig. 1f). Thus, CB1 receptor activation by both exogenous and endogenous cannabinoids leads to suppression of excitatory transmission at the ACC→DMS pathway.

The expression of the eCB system in the ACC→DMS pathway
We used RNAscope in situ hybridization to characterize the expression of genes involved in the signaling and degradation of the eCB ligand 2-AG in the ACC. 2-AG mediates DSE and is primarily degraded by MAGL in or near the presynaptic terminal where it activates CB1 receptors40. We investigated the expression of Cnr1 (encoding CB1) and Mgll (encoding MAGL) in the two major neuron types in the ACC. Excitatory ACC neurons expressed mRNA for the vesicular glutamate transporter 1 (Vglut1), while inhibitory ACC neurons expressed mRNA for the vesicular GABA transporter (Vgat, Fig. 1g). Cnr1 was expressed on ~80% of excitatory and inhibitory ACC neurons (Fig. 1h). Mgll was expressed in all somata of excitatory and inhibitory neurons; Mgll puncta density was not significantly different between glutamatergic and GABAergic neurons (Supplementary Fig. 2). Overall, these data demonstrate that the presynaptic components of the eCB system are present in ACC neurons.

2-AG is synthesized from diacylglycerol in dendrites by diacylglycerol lipase α (DAGLα)41. The striatum contains two major neuronal populations: MSNs expressing D1 (Drd1) or D2 (Drd2) receptors. RNAscope showed that Drd1 or Drd2 expressing neurons were present in approximately equal proportions. DAGLα mRNA (Dagla) was ubiquitously expressed in both Drd1 and Drd2 MSNs, with no significant differences in expression density between the two neuronal populations (Supplementary Fig. 3). Thus, the DMS expresses Daglα and can synthesize the eCB 2-AG, as shown by the presence of DSE (Fig. 1e).

eCBs are released by DMS neurons in vivo and activate CB1 receptors on ACC terminals
We used GRAB eCB2.0, a GPCR-based fluorescent sensor with low micromolar sensitivity to eCBs42, to investigate eCB dynamics at the ACC→DMS synapse in freely moving mice. AAV9-hSyn-eCB2.0 was injected into the ACC, and an optical fiber was implanted into the DMS to record eCB transients on ACC terminals (Fig. 2a, b). We first validated the efficacy of GRAB eCB2.0 to detect CB1 receptor activation using WIN 55212-2 (5 mg/kg i.p.). WIN 55212-2 produced a robust increase in GRAB eCB2.0 fluorescence within minutes of administration which was blocked by pretreatment with the CB1 receptor antagonist AM251 (10 mg/kg i.p, Supplementary Fig. 4a-d). JZL-184 is an irreversible and highly selective MAGL inhibitor that enhances extracellular concentrations of 2-AG43. Spontaneous eCB transients were relatively sparse at baseline, administration of JZL-184 (20 mg/kg, i.p.) led to an increase in the frequency of eCB transients at ACC terminals (Supplementary Fig. 4e, f). Injection of AM251 (10 mg/kg, i.p.) 20 minutes prior to JZL-184 blocked the eCB transients (Supplementary Fig. 4e, f). Thus, exogenous and endogenous cannabinoid signaling can be measured using GRAB eCB2.0 at ACC terminals in the DMS in vivo.

eCB signaling at the ACC-DMS synapse changes dynamically during approach and avoidance behavior
The DMS is involved in organizing locomotor activity44, action sequences45, and behavioral avoidance46. To identify behavioral conditions that trigger eCB release at ACC→DMS synapses, we determined whether GRAB eCB2.0 signals change as mice transition into anxiogenic areas. Mice are naturally averse to open spaces and bright lights such as the center of an open field. GRAB eCB2.0 fluorescence increased as mice entered the center zone and was accompanied by locomotor acceleration (Fig. 2c, d). Mice reached an average speed of ~100mm/sec as they entered the center zone. However, the general onset of comparable speed (100 mm/sec) in the open field was not associated with a change in GRAB eCB2.0 fluorescence (Fig. 2e). Therefore, the elevation in GRAB eCB2.0 signal was likely an effect of entering the center zone rather than locomotor initiation.

The elevated plus maze contains discrete transitions between open and closed arms. We next investigated eCB changes during key transitions in the elevated plus maze. GRAB eCB2.0 signal increased as mice approached and exited the closed arms, peaked during entry into the open arms, and declined rapidly upon re-entry into closed arms (Fig. 2f-j). If mice chose to enter the open arm rather than the closed arm, GRAB eCB2.0 fluorescence briefly remained elevated above baseline levels (Fig. 2h). In contrast, transitions from the open arm to a closed arm were associated with a reduction in GRAB eCB2.0 fluorescence (Fig. 2i, j). These findings indicate that the eCB signaling increases at ACC terminals in the DMS as mice approach anxiogenic areas and decreases as mice leave anxiogenic areas.

eCB signal increases at ACC terminals in the DMS after exposure to aversive stimuli but not conditioned cues
GRAB eCB2.0 fluorescence was recorded from ACC terminals in the DMS during fear conditioning (tone and shock pairing) and during recall (tone alone) one day later (Fig. 3a). During conditioning, mice were presented with four 20 s tones (80 dB), each co-terminating with a 1 s foot shock (0.5 mA) and separated by variable inter-trial intervals (60-80 s). ACC terminals exhibited a robust increase in GRAB eCB2.0 fluorescence following foot shock delivery (Fig. 3b), but not at tone onset (Fig. 3c). When the conditioned tone was presented alone to mice in a novel context 24 hours later, no significant changes in GRAB eCB2.0 fluorescence were observed at either the tone onset or offset (Fig. 3d, e).

Next, we determined whether exposure to a predator odor alters GRAB eCB2.0 fluorescence at ACC terminals in the DMS. The synthetic predator odor 2-methyl-2-thiazoline (2-MT) elicits freezing and avoidance behaviors in mice47. Mice were first acclimated to a novel home cage and then exposed to a control water odor, followed 20 minutes later by 2-MT (Fig. 3f). Introduction of 2-MT, but not water, produced a significant increase in GRAB eCB2.0 fluorescence at ACC terminals in the DMS (Fig. 3g, h). Together, these results indicate that unconditioned aversive stimuli, including foot shock and predator odor, elevate eCB signaling at the ACC→DMS synapse.

Genetic deletion of the CB1 receptor in ACC→DMS neurons induces anxiolytic-like behavior
The above results suggest that eCB signaling is elevated at the ACC→DMS synapse as mice transition from areas perceived to be safe to those that are dangerous. The elevation of eCBs at ACC terminals may serve as either an anxiogenic signal that promotes avoidance or a mechanism to allow the mouse to cope with perceived threat. To examine the valence of CB1/eCB signaling at the ACC→DMS synapse, we investigated how selective genetic deletion of the CB1 receptor in ACC neurons that project to the DMS altered anxiety-like behavior. Knockout of the CB1 receptor in ACC→DMS projection neurons was accomplished by injecting AAVrg-EF1a-mCherry-IRES-flpo into the DMS and a mixture of AAV9-EF1a-fDIO-Cre and either AAV9-FLEX-EGFP (for behavioral studies) or AAV9-DIO-ChR2-EYFP (for validation of CB1 receptor deletion) into the ACC of either Cnr1LoxP/LoxP or Cnr1WT/WT mice (Fig. 4a-d). In principle, Flpo is expressed in all DMS afferents, but Cre expression is limited to ACC→DMS neurons, enabling CB1 receptor deletion and eGFP labeling specifically in projection-defined ACC neurons. Six weeks after mice received the viral injections, we performed slice electrophysiological recordings of DMS neurons evoking opto-EPSCs in ACC axons that project to the DMS. Bath application of WIN 55212-2 (4 µM) suppressed opto-EPSCs in Cnr1WT/WT mice, but not Cnr1LoxP/LoxP mice, suggesting that the CB1 receptor was deleted in ACC neurons that project to the DMS (Fig. 4b). Moreover, we found that AAV9-FLEX-EGFP was expressed in ACC neurons when AAVrg-EF1a-mCherry-IRES-flpo was injected into the DMS and AAV9-fDIO-Cre was injected into the ACC (Fig. 4d). In contrast, EGFP was not expressed when flpo was omitted regardless of whether fDIO-Cre was present or not. Together these results suggest that Cre expression in the ACC depends on the presence of flpo and this approach results in genetic deletion of the CB1 receptor from ACC neurons that project to the DMS.

After ~6 weeks of AAV expression, mice underwent a battery of behavioral tests to assess anxiety- and depression-like behavior (Fig. 4c). In the open field test, mice with ACC→DMS pathway-specific deletion of CB1 receptor (ACC→DMSCB1cKO) and controls (ACC→DMSWT) spent little time in the center during the first 2 minutes of the test. However, ACC→DMSCB1cKO mice rapidly increased the time spent in the center of the open field, an effect was sustained throughout the remainder of the test (Fig. 4e-g). Although ACC→DMSCB1cKO mice exhibited reduced total distance traveled during the open field test, their initial locomotor activity (Fig. 4h,i) and maximal speed (Supplementary Fig. 5) were not significantly different from controls. The subsequent reduction in locomotion is therefore unlikely to reflect motor impairment and is more consistent with faster habituation to the novel environment. In the elevated plus maze (EPM), ACC→DMSCB1cKO mice spent significantly more time in the open arms compared to ACC→DMSWT mice (Fig. 4j, k). There was no significant genotype difference in the number of open arm entries, center entries or time spent in the center zone (Fig. 4l-n). ACC→DMSCB1cKO mice had reduced overall distance traveled and speed compared to ACC→DMSWT mice (Supplementary Fig. 6). This was primarily due to reduced speed of ACC→DMSCB1cKO mice in the closed arms, rather than the open arms (Supplementary Fig. 6).

The novelty-suppressed feeding test assesses avoidance behavior in a conflict. The fasting mouse faces a dilemma between feeding and avoiding open space within the novel environment. A reduction in the latency to feed indicates that hunger is weighted more heavily than avoidance48. ACC→DMSCB1cKO mice had a reduced latency to feed compared to ACC→DMSWT mice (Fig. 4o). Mice defensively bury neutral objects, such as marbles, in response to anxiogenic environmental stimuli to reduce stress49. Stressed mice bury more marbles, while anxiolytic drugs reduce the tendency to bury marbles49, 50. In the marble bury test, ACC→DMSCB1cKO mice buried fewer marbles compared to ACC→DMSWT mice (Fig. 4p). Together, these results show that loss of CB1 from ACC neurons that project to the DMS produced anxiolytic-like effects.

Interestingly, we did not observe any difference between genotypes in the sucrose preference test (Supplementary Fig. 7) or forced swim test (Fig. 4q). Moreover, conditioned freezing responses to tone did not differ between genotypes during fear conditioning and extinction (Supplementary Fig. 8a-c). Overall, these results indicate that ACC→DMSCB1cKO mice had an anxiolytic phenotype, did not display anhedonia or behavioral despair, and retained the ability to form associations between environmental cues and aversive stimuli.

CB1 receptor activation constrains the appetitive and anxiolytic effects of optogenetic stimulation of the ACC→DMS pathway
Real-time place preference (RTPP) or aversion (RTPA) is commonly used to assess reinforcing or aversive effects of optogenetic stimulation of specific circuits51. We tested whether in vivo optogenetic stimulation of the ACC→DMS pathway was reinforcing or aversive in ACC→DMSWT or ACC→DMSCB1cKO mice with a RTPP/RTPA paradigm (Fig. 5a-d). Compared to baseline, optogenetic stimulation of the ACC axon terminals with blue light (20 Hz, 465 nm) produced no significant change in preference or distance traveled in ACC→DMSWT mice but produced RTPP in ACC→DMSCB1cKO mice without altering the distance traveled (Fig. 5d-f). These findings reveal a CB1-dependent constraint on the appetitive impact of ACC→DMS activation. Next, we assessed whether in vivo optogenetic stimulation of the ACC axon terminals in the DMS could alter anxiety-like behavior in ACC→DMSWT or ACC→DMSCB1cKO mice using the open field and elevated plus maze. In the open field test, there were no significant differences in distance traveled or time spent in the center between genotypes during the optogenetic stimulation period (Supplementary Fig. 9a, b). In the elevated plus maze, ACC→DMSCB1cKO mice spent significantly more time in the open arms and center zone compared to ACC→DMSWT mice (Fig. 5g, i). The number of open arm and center zone entries during the stimulation period were also greater in ACC→DMSCB1cKO mice compared to ACC→DMSWT mice (Fig. 5h, j). These data indicate that CB1 deletion at the ACC→DMS pathway unmasks a shared circuit mechanism whereby its activation can induce both place preference and anxiolysis.

Aversion to THC requires CB1 receptor expression on ACC neurons that project to the DMS
High doses of THC reliably produce feelings of anxiety in humans32, 52, 53 and avoidance behavior in rodents54, 55. Our data thus far suggest that eCB signaling at the ACC terminals in the DMS precipitates avoidant behavior in response to aversive stimuli and may therefore be a locus where activation of the CB1 receptor by exogenous agonists, such as THC, promotes avoidance. High-dose THC conditioned place aversion (CPA)54, 55 was examined in ACC→DMSWT and ACC→DMSCB1cKO mice. Mice were first tested for basal place preference in a two-chamber apparatus (pre-test). For the next five days, mice were given an injection of high dose THC (25 mg/kg i.p.) in the preferred chamber and a vehicle injection in the less preferred chamber. The final test was conducted drug-free, which minimized potential confounds from THC-induced motor suppression on CPA expression (Fig. 6a, b). In the CPA trial, ACC→DMSWT showed a marked reduction in the time spent in the THC-paired chamber and a corresponding increase in time spent in the vehicle-paired chamber on the test day compared with the pretest day, suggesting that THC-pairing induces CPA. In contrast, ACC→DMSCB1cKO showed no significant difference in the time spent in either the THC-paired chamber or vehicle-paired side between pretest and test days (Fig. 6c, d; Supplementary Fig. 10a). Locomotor activity on the test day was reduced compared with the pretest day, likely reflecting habituation to the apparatus rather than drug effects (Supplementary Fig. 10b). Thus, deletion of CB1 in ACC→DMS neurons attenuates THC-induced CPA.

Methods
Animals
Animal use and maintenance was in accordance with protocols approved by the Institutional Animal Care and Use Committee of The Medical College of Wisconsin. Mice were given ad libitum access to food and water, unless stated otherwise, and housed four to five per cage in a temperature (23 ± 1°C) and humidity-controlled room (40–60%) with a 14 hr light, 10 hr dark cycle. All experiments were performed on adult male and female mice (8–16 weeks old at the start of experiments). Roughly equal numbers of male and female mice were included in all experiments. Both sexes exhibited similar responses, data from both sexes were pooled. Further disaggregation of data by sex was not statistically powered for detecting sex difference due to the sample sizes. C57BL/6J (Jax stock#: 000664) were obtained from the Jackson Laboratory (Bar Harbor, Maine). Cnr1loxP/loxP mice (Cnr1tm1.1Zxx, MGI: 6119514) were generated66 and maintained on a C57BL/6J background.

Stereotaxic surgery and AAV injection
Mice were anesthetized by isoflurane inhalation prior to surgery. One hole was drilled above each injection site. For all AAV injections into the ACC 150 nl of total volume was injected at AP: +0.98, ML: ± 0.28, DV: 1.45-1.5. For all AAV injections into the DMS 300 nl of total volume was injected at AP: +0.98, ML: ± 1.35, DV: 2.65-2.7. For electrophysiological experiments, AAV5-CaMKIIa-hChR2(H134R)-mCherry (Addgene #26975) was injected into the ACC. Mice were allowed about 2 weeks to recover before electrophysiology experiments. For pathway specific knockout behavioral experiments, AAVrg-EF1a-mCherry-IRES-Flpo (Addgene # 55634) was bilaterally injected into the DMS. After DMS injections, a 1:1 cocktail of AAV9-EF1a-fDIO-Cre (Addgene #121675) and AAV9-EF1a-FLEX-EGFP (Addgene #98747) or AAV9-DIO-Chr2-EYFP (Addgene #35509) was injected into the ACC. Animals were housed individually following surgery. Experiments were performed 6 weeks after these injections to allow for genetic knockout.

For fiber photometry experiments, AAV9-hSyn-eCB2.0 (WZ Biosciences, Columbia, MD) was injected into the ACC. Next, a low autofluorescence fiber-optic cannula (OD = 200 µm, NA = 0.57; Doric Inc, Québec, Canada) was implanted into the DMS 2.6 mm below the surface of the skull and 1.35 mm to the right or left of the bregma. For in vivo optogenetic experiments two low autofluorescence cannulae were implanted into the DMS at a 10-degree angle 2.6 mm below the skull surface and 1.6 mm to the right and left of the bregma. Two screws (00–96 × 1/16; Invivo1) were screwed into the skull and held in place by C&B Metabond quick adhesive cement to secure the cannula in place.

Behavior
Sample sizes for behavioral tests were calculated based on statistical power analysis (α = 5%, Power = 0.8), with consideration of prior experience and sample sizes generally used in the field. For tests that required manual scoring (e.g. forced swim test and marble burying test), the experimenters were blinded to groups. The least stressful behavioral tests were performed before high stress tests. Tests were performed in the order listed below and no more than one behavioral test was performed in a single day.

Open field test (OFT): Mice were placed in one corner of an open field (50 cm × 45 cm × 30 cm) and were allowed to explore for 20 minutes. Locomotor activity was recorded with ANY-maze (Stoelting, Wood Dale, IL). The total distance traveled, number of center entries and time spent in the center was calculated. The center zone was defined as the central 25 cm × 22.5 cm area of the box. For fiber photometry experiments, Danse (Doric Lenses Inc.) was used in place of ANY-maze.

Elevated plus maze (EPM): The EPM was elevated 40 cm above the floor. Mice were placed in the center zone and allowed to explore the maze for 5 minutes. For fiber photometry experiments utilizing the EPM, mice were allowed to explore longer (15 minutes). The mice were tracked with ANY-maze. The time spent in the open arms and the number of entries was quantified. For fiber photometry experiments Danse was used to track animals.

Novelty-suppressed feeding (NSF): Mice were deprived of food for 24 hours before being placed in a novel environment (a plastic box 50 cm long × 35 cm wide × 30 cm deep). One pellet of regular chow was placed in the center. A mouse was placed in the corner of the box and the latency to feed was determined. Feeding was only counted if mice bit with the use of their forepaws, rather than sniffing or bumping into the food. Immediately following the test, the latency to feed in the home cage was measured.

Marble burying test (MBT): Mice were placed in a cage filled with 7–8 cm of bedding. Twenty-four dark blue marbles (1.35 cm diameter) were on the top of the bedding. Mice were allowed to freely in the cage for 20 min. At the end of the 20 min session, the marbles buried (2/3 covered or more) were counted.

Sucrose preference (SP): Mice were habituated to drinking from a single bottle for at least 48 hours before testing. Eight hours prior to the SP test, food and water were removed. During testing, two bottles were placed in the home cage: one containing 1% sucrose solution and the other containing plain tap water. After 16 hours, sucrose preference (%) was calculated as the mass of sucrose solution consumed divided by the total mass of fluid consumed.

Forced swim test (FST): Mice were placed into glass cylinders (13 cm diameter, 25 cm tall) filled with ~18 cm of water (30 ± 1°C). The mice were left in the cylinders for 6 min and the time spent immobile during the last 4 min of the test was scored. Immobility was defined as the absence of all movements except those required to keep the mouse’s head above the water.

Fear conditioning and extinction: Fear conditioning and extinction training were performed in light and sound attenuating boxes (Actimetrics, Wilmette, IL, USA). FreezeFrame 5.0 software was used to measure freezing behavior (Actimetrics). During fear conditioning, mice were exposed to 5 auditory tones (75–85 dB, 20 s) that co-terminated with a 1 second foot shock (0.5 mA). Intertrial intervals were 60–80 s between the cues. Fear extinction training began one day later. Mice were exposed to ten 20 s tones in a novel environment with 60–80 s pseudorandom intervals without foot shocks. This procedure was repeated every 24 hours for 10 days.

Conditioned place avoidance (CPA): During the pre-test, mice were placed in a two-chamber conditioned place preference apparatus for 30 minutes. One chamber featured vertical stripes and the other dots, with illumination maintained at 5-10 lux in each side. Time spent in each chamber was recorded to determine initial preference. For the subsequent five days, mice underwent place conditioning. On each day, mice received a vehicle injection in their initially less-preferred chamber and, four hours later, received THC (25 mg/kg, i.p.) in their preferred chamber. Immediately after each injection, mice were confined to the corresponding chamber for 45 minutes. THC was dissolved in ethanol, and both THC and vehicle solutions were prepared at a 1:1:18 ratio of ethanol:kolliphor:saline.

Slice preparation and electrophysiology
Artificial cerebral spinal fluid (ACSF) contained the following (in mM): 119 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, and 10 glucose. Choline-based solution contained the following (in mM): 110 choline chloride, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgSO4, 26 NaHCO3, 25 glucose, 11.6 sodium ascorbate and 3.1 sodium pyruvate (pH to 7.4). Prior to slice cutting, mice were anesthetized by isoflurane inhalation and received transcardial perfusion of a 10 ml mixture of artificial cerebral spinal fluid (40%) and choline based cutting solution (60%). We found that transcardial perfusion with this solution improved DSE responses in the striatum. Following transcardiac perfusion mice were decapitated and immediately embedded in 3% low-gelling temperature agarose at 40˚C in a cylindrical steel mold. The mold was immediately transferred to ice and left to incubate for 2 minutes. After the agarose solidified, the mold was trimmed to reveal prefrontal cortex. Brain slices (250 µm) containing the ACC and DMS were cut on a vibratome (Leica VT1200s) in choline-based solution before being transferred to a separate beaker containing choline cutting solution. ACSF was gradually introduced into the beaker before the slices were transferred to ACSF. Slices were incubated in ACSF for 30 minutes before recording. Cutting steps were performed at room temperature. All solutions were saturated with 95% O2 and 5% CO2.

Whole-cell patch clamp recordings were made from DMS neurons using patch-clamp amplifiers (Multiclamp 700B) under an infrared differential interference contrast microscope67, 68. Data acquisition was performed using DigiData 1440A and 1550B digitizers and analysis software pClamp 10.7 (Molecular Devices). Signals were filtered at 2 kHz and sampled at 10 kHz. Neurons were voltage-clamped at −80 mV unless stated otherwise. Optical stimulation was delivered through a 40x objective lens via Lumencor Spectra X light engine. For whole-cell recordings of opto-DSE, glass pipettes (4–6 MΩ) were filled with an internal solution containing the following (in mM): 140 K-gluconate, 8 NaCl, 10 HEPES, 0.4 EGTA-Na, 2 MgCl2, 2 Mg-ATP, 0.3 Na GTP, pH 7.2 with KOH. With this slicing procedure, there was substantial run up of opto-EPSCs, especially during the first 10 minutes of whole cell recording. DSE was not recorded until neurons reached a stable baseline for opto-EPSCs, which was ~10-15 minutes after breaking in. Series resistance (10–30 MΩ) was monitored throughout all whole-cell recordings, and data were discarded if the resistance changed by > 20%. All recordings were performed at room temperature.

RNAscope
The RNAscope protocol used can be found elsewhere66. Briefly, mice were deeply anesthetized with isoflurane and transcardially perfused with 0.1 M sodium phosphate buffered saline (PBS) followed by 4% paraformaldehyde in 4% sucrose-PBS (pH 7.4). After perfusion, the brain was removed and post-fixed in the same fixative for 2 hr at 4°C and was then dehydrated in increasing concentrations of sucrose (20% and 30%) in 0.1 M PBS at 4°C and frozen on dry ice. Coronal sections (14 μm) were cut with a Leica cryostat (Leica CM1860) and mounted on Superfrost Plus slides (Fisher Scientific). Probes targeting the mRNA transcripts of Daglα (target region: base pairs 396–1259 of Mus musculus diacylglycerol lipase alpha, NM_198114.2), Drd1 (target region: base pairs 444–1358 of Mus musculus dopamine receptor D1, NM_010076.3), Drd2 (target region: base pairs 69–1175 of Mus musculus dopamine receptor D2 NM_010077.2), Cnr1 (target region: base pairs 530–1458 of Mus musculus cannabinoid receptor 1, NM_007726.3), Slc17a7 (target region: base pairs 498–1009 of Mus musculus solute carrier family 17 (vesicular glutamate transporter) member 7 (Slc17a7), NM_182993.2), and Slc32a1 (target region: base pairs 894–2037 of Mus musculus solute carrier family 32 (GABA vesicular transporter) member 1 (Slc32a1), NM_009508.2), were designed by and purchased from Advanced Cell Diagnostics Inc. (Hayward, CA). The manufacturer’s instructions for the RNAscope Multiplex Fluorescent V2 Assay were followed. Slides were counterstained with DAPI and mounted with Vectashield Antifade Mounting Medium (Vector Laboratories) and imaged on a Leica TCS SP8 confocal microscope with a 25x/0.95 NA/2.4 mm WD HC FLUOTAR water immersion objective. Imaris (Bitplane) was used for quantifying the imaging data. For analysis, cell-specific markers (Drd1 vs. Drd2; Slc17a7 vs. Slc32a1) were reconstructed as surfaces in Imaris. The Spots function was then applied to detect Dagla and Mgll puncta using a threshold of 1 μm diameter. The number of fluorescent puncta was quantified using the Spots tool.

In vivo fiber photometry of GRAB eCB2.0
A fiber photometry system (Doric Lenses, Inc) was used to detect eCB-dependent changes in fluorescence from GRAB eCB2.0 in the DMS of freely moving mice69. In brief, a computer-controlled fiber photometry console and low-power LED drivers (LEDD_4) generated 405 and 465 nm light from LEDs. LED intensities were sinusoidally modulated by a lock-in amplifier at nonsynchronous frequencies (208.616 and 572.21Hz for 465 and 405 nm, respectively) to filter out the fluorescence arising from excitation spectra. A fluorescence MiniCube (Doric; FMC6_IE(400-410)_E(460-490)_F(500-550)_ E(555-570)_F(580-680)_S) directed and combined the excitation lights through a fiber-optic patch cord (Doric; MFP_200/230/900–0.57_1.0_FCM_ZF1.25_LAF) and rotary joint (Doric; FRJ_200/230/LWMJ-0.57_1m_FCM_0.15_FCM). The implanted fiber-optic cannula was connected to the patch cord by a 1.25 mm mating sleeve (Doric; 1.25 mm with black cover; Sleeve_ZR_1.25-BK). Fluorescence emissions returned through the same patch cord and were separated in the MiniCube before being redirected to photoreceivers (Model 2151, DC low setting; Newport Corporation, Irvine, CA). Time locked video was captured by an overhead camera and recorded through Doric Neuroscience Studio during fiber photometry recordings.

Prior to experiments, the patch cord was photobleached with 405 and 465 nm light for at least one hour to reduce autofluorescence from the fiber-optic cable. The power output was adjusted to ~45-55 µW at the tip of the patch cord before each recording. Data was acquired through Doric Neuroscience Studio software at a rate of 12.0 ksps, before being decimated by a factor of 100. After the patch cord was attached, mice were allowed to acclimate in their home cage for 15 min to allow environmental and patch cord habituation before the start of the experiment. Behavioral tests were performed at one-week intervals prior to pharmacological tests. All animals received an injection of WIN 55212-2 after completion of all behavior experiments to validate signal. The open field test was 10 minutes. The elevated plus maze test was 15 minutes.

For open field and elevated plus maze tests, mouse movement and zone transitions were automatically detected using Danse. For the elevated plus maze, a baseline period of −20 to −15 s was selected because mice were typically positioned away from the center or open arms at this point. This baseline (−20 to −15 s) was compared with the 5 s preceding a compartment transition (pre-event: −5 to 0 s) and the periods directly after a compartment transition (post-event 1: 0 to 5 s; post-event 2: 5 to 10 s).

For fear conditioning and recall, mice were trained in a Med Associates (St. Albans, VT) operant chamber. After a 2-minute habituation period, mice were presented with a 20-second tone (4 kHz) that co-terminated with a 1-second, 0.5 mA foot shock. This tone–shock pairing was repeated four times with a randomized inter-trial interval of 60–80 s. Twenty-four hours later, fear recall was assessed in a novel context in which mice received tone presentations without shock. A Med Associates console sent TTL signals for tone and shock to Doric console that were used for peri-event analysis.

For 2-MT exposure experiments mice were placed into a new home cage without bedding and allowed to acclimate for 20 minutes. Then, a small glass container, which held a piece of filter paper with 20 l of ddH2O was placed in the home cage. After 20 minutes, an identical container which held a piece of filter paper with 20 l of 2-MT (diluted 1:50 times in ddH2O) replaced the container holding only ddH2O.

Following the completion of the experiments, AAV expression in the ACC, and the optic fiber implants in the DMS were verified by post hoc imaging on a VS200 slide scanner (Evident Scientific).

Analysis of fiber photometry results
The fiber photometry data were analyzed using Doric Neuroscience Studio and Danse software (Doric Inc, Québec, Canada). For Z-score generation, a running average of 2 seconds was used to smooth the raw 465 nm and 405 nm traces. The z-scored ΔF/F values were calculated by applying an adaptive iteratively reweighted Penalized Least Squares (airPLS) fit to the data70, the fitted 405 nm signal was subtracted from the fitted 465 GRAB eCB2.0 signal and then divided by the fitted 405 nm signal. Location of the mouse, speed, and zone entries and exits were tracked in Danse. Peri-event analysis of z-scored values and area under the curve was performed with the “peri-event” function in Danse. For GRAB eCB2.0 transient frequency analysis, data were exported to MiniAnalysis.

In vivo optogenetics during behavior
Following AAV injections and fiber optic cannula placement (200 m core, 0.5 NA), mice were allowed to recover for 6 weeks for viral gene expression and genetic knockout to occur. Mice were acclimated to the optical patch cord for 10 minutes for two days (20 min total) before the start of behavioral experiments. Behavioral measurement and control of in vivo optogenetic stimulation was coordinated through ANY-Maze. For optogenetic experiments, mice received 20 Hz pulses (5 ms on 45 ms off) of 473 nm laser stimulation from the ANY-maze controlled computer which were converted to TTL signals via and AMI-2 optogenetic interface (Wood Dale, Illinois) and sent to a DPSSL laser system (Laserglow, Toronto, Ontario). Laser intensity was measured at ~4 mW at the fiber tip. For RTPP, mice were placed in a 27 cm x 51 cm apparatus bisected into two chambers with equal area connected by a 10 cm wide opening. Mice were given a 10-minute baseline to acclimate to the chamber prior to testing. Then, mice underwent a 10-minute period where optical stimulation occurred in one side of the apparatus, but not the other. The percent time spent on the stimulation side and distance traveled was compared during the stimulation phase to the baseline. For the open field test mice were placed in the chamber for a 2-minute baseline that was followed by a 2-minute stimulation on period and a 2 minute off period. For the elevated plus maze, mice underwent a 2-minute baseline followed by a 2-minute stimulation on period and a 2-minute stimulation off period.

Chemical reagents
AM251 and WIN 55212-2 were purchased from Tocris Bioscience (Ellisville, MO). JZL-184 was purchased from Cayman Chemical. 9-Tetrahydrocannabinol (THC) was provided by the NIDA Drug Supply Program. All other common chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Statistics
Data are presented as the mean ± s.e.m.. Animal numbers and sample sizes are calculated based on statistical power analysis (α = 5%, Power = 0.8). For behavioral tests, variance and normality were assessed prior to statistical testing with SigmaPlot. Data sets were compared with either Student’s t-test, Paired t-test, Kaplan-meier survival analysis, one-way ANOVA or two-way repeated measures (RM) ANOVA followed by Tukey’s, Bonferroni, or Holm-Sidak post hoc analysis. Post hoc analyses were performed only when ANOVA yielded a significant main effect or a significant interaction between the two factors. Results were significant at p < 0.05.

Data Availability
All data supporting the findings of this study are included in the main article and supplementary materials. Source data are provided within the article. Processed data generated in this study have been deposited in FigShare. Statistical details and conclusions are reported in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a well-executed, innovative, exciting and interesting paper describing the role of CB1 on ACC-DMS glutamatergic neurons and their role in the regulation of anxiety and aversion induced by high dose THC. The work would be of high interest to the field. The work is provocative since CB1 on glutamatergic forebrain neurons is thought to primarily mediate the anxiolytic effects of cannabinoids and exert anti-anxiety effects. The paper therefore identified a very discrete sup-circuit which produces the opposite effects. The paper is clear, easy to read, and the figures are well-presented. The experiments appear of high quality. However, I have some concerns that relate to the strength of the conclusions. The primary of which is the unanswered question of whether the ACC-DMS pathway directly regulates place preference/aversion and the aversive effects of THC or are CB1 on ACC-DMS terminals able to reduce THC aversion indirectly by reducing the anxiogenic effects of high dose THC. This issue needs to be resolved experimentally (see #3-4 below).\nMajor concerns:\n1) Figure S1 does not make sense. How can 80% of GABA neurons express CB1. CB1 is confined to a small subset of GABA cells in the cortex. This likely represents a technical artifact.\n2) Figure 2 should be supplemental as it is just tool validation and does not really add much to the main paper, and replicates other work showing sensor increases to CB1 direct and indirect agonists.\n3) A major conclusion of the work is that CB receptors constrain an anxiolytic ACC-DMS circuit, and these receptors are required for THC aversion. For the latter conclusions to be fully supported, the authors need to show 1) that activation of the ACC-DMS pathway produces place preference (using RTPP assay) and 2) that CB deletion increases glutamate release at AAC-DMS synapses or the in vivo activity of this pathway during place preference expression. Ideally (although understandably harder), CB1 deletion from this pathway should turn subthreshold optogenetic place preference into suprathreshold place preference. Without these data, there is no link between the EPM/open field data (behavior and sensor) and THC aversion data.\n4) It is possible that ACC-DMS will not produce place preference alone. This would suggest that reduced THC-induced anxiety underlies the reduced place aversion. Showing that AAC-DMS CB1 deletion blocks increased anxiety induced by high dose THC would be needed to support this alternative hypothesis. Doing these studies first might preclude the necessity for #3 if this deletion clearly blocks the anxiogenic effects of high dose THC, in which case the conclusions would be that blocking anxiogenic effects of THC results in secondary reduced THC aversion.\n5) Changes in sensor signal after marble burying are not convincing, just as many bouts of increased activity are seen on the dot-plot. i.e. the magnitude is very small and biological significance is questionable.\nMinor concerns:\n1) The AUC data for cannabinoid sensor is hard to follow. May be best to label the baseline, pre-exit, post exit time periods on the time course data so the readers know where the AUC measures are being taken.\n2) The negative fear conditioning data could also be a supplement\n3) There is no functional dissociation of ACC to D1/D2 pathways, this at least needs to be noted as a limitation of the work. It should also be placed in context of cannabinoid plasticity segregation/specificity at cortical to D1 and D2 synapses (i.e. work of Lovinger, Melenka and others). Also ref 30 indicates asymmetric innervation of D1 over D2 neurons in the DMS.\n4) Number of biological replicates is not shown for Figure 3. The number of transitions is used for the “n” for statistical analysis but without knowing how many mice this is derived from the rigor is hard to evaluate.\n5) Do AAC-DMS neurons collateralize to innervate other targets? This might change the interpretation of the circuit-specific KO data.", "reviewer_2": "This study investigated the role of CB1 receptor signaling in the anterior cingulate cortex (ACC) to dorsomedial striatum (DMS) pathway in regulating anxiety-like behavior and aversive responses to cannabinoids. Key findings include that CB1 receptors and enzymes involved in 2-AG synthesis and degradation are detected in the ACC and DMS. Optogenetic electrophysiology demonstrates that cannabinoids suppress excitatory transmission from ACC to DMS neurons. In vivo fiber photometry reveals increased endocannabinoid release in the DMS following administration of WIN55,212-2 or exposure to risk conditions (open arm of EPM, center of open field). Genetic deletion of CB1 in ACC→DMS neurons reduces innate anxiety-like behaviors and prevents THC-induced conditioned place aversion. The authors concluded that CB1 receptor activation in this circuit contributes to the anxiogenic effects of cannabinoids, providing insights into cannabis-induced aversive effects. The study integrates advanced methodologies, including in vivo fiber photometry, optogenetic electrophysiology, and behavioral assays, to present a well-supported conclusion. These results offer novel insights into how endocannabinoid signaling in the ACC→DMS pathway influences anxiety and aversion, addressing a significant gap in cannabinoid research.\nMajor concerns:\n• The title should better reflect the key findings regarding the role of ACC→DMS CB1 receptor signaling in anxiety-like behavior.\n• The introduction should include a more comprehensive background review on the dose-dependent effects of cannabinoids, highlighting their anxiolytic and rewarding effects at low doses and anxiogenic and aversive effects at high doses. Additionally, the involvement of other brain regions such as amygdala, BNST, and NAc should be cited.\n• ACC glutamatergic neurons likely project to multiple brain regions, a supplementary figure showing these projections would clarify the significance of the ACC→DMS pathway in this study.\n• The images showing Dagla expression in D1- and D2-MSNs (Fig. 1g) and Mgll expression in ACC GABAergic and glutamatergic neurons (Extended data Fig. 2) are unclear. Higher-magnification images and more detailed methods for identifying these neurons would improve the clarity of these results. If possible, quantifying the percentage of ACC or DMS neurons expressing Dagla or Mgll would strengthen the conclusions.\n• According to the retrograde endocannabinoid signaling hypothesis, CB1 activation reduces eCB (2-AG) release (Fig. 2g), yet WIN55,212-2 induces a robust increase in eCB signaling via a CB1-dependent mechanism (Fig. 2c). This apparent contradiction should be addressed.\n• WIN55212-2 was used in the eCB signal assays, while THC was used in the CPA experiments. It would be valuable to determine whether THC similarly increases eCB release in the DMS as WIN55212-2 does.\n• The authors claim that eCB signaling increases as mice exit closed arms and enter open arms of the EPM and decreases in the opposite transition. However, data in Fig. 3 (d, f, e, g) suggest that GRABeCB2.0 signals similarly decline after both closed and open arm exits or entry. Further clarification and discussion of these findings are needed.\n• While Extended Data Fig. 5 provides electrophysiological evidence of CB1 deletion, additional IHC or RNAscope images confirming CB1 loss specifically in ACC→DMS neurons would be beneficial. If unavailable, Extended Data Fig. 5 should be relocated to the main text.\n• The behavioral data in Fig. 4e and Fig. 4k appear inconsistent and should be clarified.\n• The 25 mg/kg THC dose used in the CPA experiment is quite high and could induce sedation, hypothermia, or motor impairments, potentially confounding place preference results. A lower dose should be tested, or at minimum, these concerns should be addressed in the discussion.\n• The study attributes THC-induced conditioned place aversion solely to CB1 receptor activation in the ACC→DMS pathway. However, systemic THC effects on other circuits should be acknowledged in the discussion.\n• The hypothesis that \"inhibition of ACC terminals in the DMS causes anxiety-like behavior and thereby contributes to the aversive effects of THC\" does not account for how low doses of cannabinoids or THC produce anxiolytic effects. The potential involvement of other CB receptors, neuronal subpopulations, or circuits in cannabinoid-induced reward and anxiolysis should be discussed.\nMinor Points\n1. THC is not a selective CB1 receptor agonist (line 56). This should be revised for accuracy.\n2. WIN55,212-2, at 5 mg/kg, robustly increases eCB signaling in the DMS. Is this effect dose-dependent? Additional clarification would be useful.\n3. In electrophysiology and RNAscope experiments, the sample sizes refer to the number of neurons or brain sections. State the number of mice used in these experiments.\n4. Figure Relocation Suggestion: Move Fig. 1g and Fig. 5 (a, b, c) to the supplementary materials while relocating Extended Data Figs. 1, 4 and 5 to the main figures for better organization.", "reviewer_3": "Reviewer comments on Manuscript NCOMMS-25-06847-T \"CB1 receptor signaling at the cingulate-striatal circuit is aversive\"\nThis manuscript aims to understand neural mechanisms for high dose THC effects in the brain through several experiments done focusing on the dorsal medial striatum (DMS) inputs from the anterior cingulate (ACC). They use a range of behavioral experiments, slice electrophysiology, and in situ hybridization to find the role of cannabinoid receptors in mostly aversive effects of THC. Authors use optogenetics and electrophysiology to show the monosynaptic connection between ACC and DMS and the presence of CB1Rs from the ACC terminals at the DMS. Moreover, with the presence of the synthesis enzyme for endocannabinoid 2-AG (DAGLa) in the DMS (with no preference to D1 or D2 medium spiny neurons), authors showed 2-AG mediated short-term depression of glutamatergic transmission (DSE). They continued in vivo physiology recordings by using the GRABeCB2.0 sensor and showed the endocannabinoid binding increases on those terminals in freely moving mice in elevated plus maze and open field tests while mice cross through the open arms (EPM) or the center of the brightly lid arena (OF) which are mostly anxigenic for mice (innately) and often avoided. Recordings on the same terminals during fear conditioning and extinction learning only showed increased binding during shock onset but not during tone onset/offset. Using an intersectional approach, they knocked down CB1Rs on those terminals and showed mice became less anxious and avoid the high dose THC paired compartment on the conditioned place preference test.\nThis manuscript is written well in a clear language explaining the reasoning of the experiments done and discuss the results obtained without speculation. Please find questions and comments to improve the manuscript.\n1- Authors showed the involvement of the CB1Rs in the ACC terminals at DMS in avoidance of aversive/stressful events. DMS majorly plays role in goal directed behaviors and have low expression of CB1Rs. Could the low expressed CB1Rs in DMS (compared to DLS) be involved in important roles in survival behavior? Maybe authors should try the fear conditioning with a predator odor to test the idea of increased endocannabinoid signaling in innate but not learned aversive stimuli. Testing their intersectional knockout mice on the predator odor conditioning and recording the terminals during the predator odor conditioning test will solidify their findings and their proposed model and neural mechanism.\n2- Conditioned place aversion test for a high dose THC applied on multiple conditioning days before the test day brings the question on how the mice behave on 6th day. Have they determined this with tetrad tests? How is the time spent in vehicle side in this experiment? Please plot and show side by side. t-test may not be the best to test the genotype +test phase related changes, please use 2-way ANOVA.\n3- Have the authors manipulated this circuit during behavior? Activation/inhibition of ACC-DMS projections in the open field and epm tests (or now suggested predator odor conditioning test).\n4- Authors don’t show any data regarding the influence of the GABAergic interneurons in DMS that may be important in modulating avoidance of stressful/aversive stimuli. Specifically, D1R expressing MSN’s and the possible involvement of anandamide should be tested in slice electrophysiology experiments.\n5- Please be more specific in baseline periods for fiber photometry experiments.\n6- Please include a small discussion on the role of ACC-DMS neurons in itching /scratching (https://doi.org/10.1038/s41467-020-16859-5) which resembles to the reaction to excessive stress.\nOther issues related to the figures and their corresponding legends:\nFigure 1: Delete repeated words on legend line 558 ‘depolarization induced’.\nFigure 2: Move the pharmacological validation figure into supplementary figures. Instead show one of the experiments suggested above.\nFigure 3: Does the line in time 0 indicate the event start? (ie. crossing to the other compartment) Explain why the baseline is -20 to -15 s in figure 3d. Shouldn’t it be 5 s before the entry? Indicate the times for baseline, pre-entry, post entry 1 and post entry 2 into the figure legends and also to the methods.\nFigure 4: The legends and the figures shown are not consistent thus confusing to the reader, please correct the letters and delete all words (such as: left, right, center) to make it clear by assigning each figure a letter. Please make new graphs to indicate the total distance moved in each compartment (open, closed arms and center) for EPM.\nCB1R ko mice are not moving much in the open field (as reflected by total distance travelled difference in ko mice) therefore could they be staying more in the center of the open field? Same problem in the EPM time in center graph doesn’t match the heat map image. Include the velocity of the mice in each compartment of the EPM test.\nFigure 5: Indicate what comparisons are done for the statistical tests. For example, in extinction result of the two-way ANOVA comparisons are from day1 to day 6. Legend ‘c’ should be changed to ‘d’. Please discuss the variation in the THC response in the CPA test.\nExtended Figure 1: be more specific, include details about the number of mice used for the RNAScope studies. And how many sections were counted from each mouse. Were they counted from 40x images?\nExtended Figure 2: correct ‘Cnr1’ to ‘Mgll’"}, "author_response": "We thank the three reviewers for their insightful and constructive comments. In response to their critiques, we have extensively revised the manuscript and performed several new experiments, which are briefly summarized below and described in detail in our point-by-point responses to each reviewer. Responses to reviewers and major revisions to the manuscript are indicated in blue font.\n We performed real time place preference (RTPP) with optogenetic stimulation of the ACC→DMS pathway. Optogenetic stimulation (20 Hz) of ACC axon terminals in the DMS induced place preference in ACC→DMSCB1cKO mice, but not ACC→DMSWT mice (Reviewer 1, see Fig. 5). These results revealed a CB1-dependent constraint on the positive motivational impact of this corticostriatal circuit.\n Reviewer 3 asked whether we directly manipulated the ACC→DMS pathway during tests of innate avoidance (e.g. open field and elevated plus maze). In response, we examined whether optogenetic stimulation of the ACC→DMS pathway produces an anxiolytic effect in ACC→DMSWT and ACC→DMSCB1cKO mice. Optogenetic stimulation of this pathway produced an anxiolytic effect in the elevated plus maze in ACC→DMSCB1cKO mice but not ACC→DMSWT mice (Fig. 5). It is likely that anxiolysis induced by ACC→DMS stimulation may also contribute to the observed RTPP experiment proposed by Reviewer 1.\n We showed that the predator odor (2-MT) induced a robust increase in GRAB eCB2.0 fluorescence in the ACC→DMS pathway (Reviewer 3, Fig. 3). Thus, GRAB signals are eCB2.0 elevated by multiple aversive stimuli (foot shock, predator odor, open spaces).\n We performed anterograde AAV tracing and whole brain mapping to show projection targets of ACC excitatory neurons (Reviewer 2, Supplementary Fig. 1).\n We have added higher magnification images for RNAscope experiments (Reviewer 2).\n We significantly increased the animal numbers for the THC conditioned place aversion (CPA) in ACC→DMSWT and ACC→DMSCB1cKO mice (Reviewer 3, Fig. 6).\n We provided locomotor activity data in both pretest and test days of CPA trial, and speed measures for the open field and elevated plus maze performed with ACC→DMSCB1cKO and ACC→DMSWT mice (Reviewer 3, Supplementary Figs. 5, 6 and 10).\nOur point-to-point responses to reviewer critiques are listed below.\nReviewer #1 (Remarks to the Author):\nThis is a well-executed, innovative, exciting and interesting paper describing the role of CB1 on ACC-DMS glutamatergic neurons and their role in the regulation of anxiety and aversion induced by high dose THC. The work would be of high interest to the field. The work is provocative since CB1 on glutamatergic forebrain neurons is thought to primarily mediate the anxiolytic effects of cannabinoids and exert anti-anxiety effects. The paper therefore identified a very discrete sup-circuit which produces the opposite effects. The paper is clear, easy to read, and the figures are well-presented. The experiments appear of high quality. However, I have some concerns that relate to the strength of the conclusions. The primary of which is the unanswered question of whether the ACC-DMS pathway directly regulates place preference/aversion and the aversive effects of THC or are CB1 on ACC-DMS terminals able to reduce THC aversion indirectly by reducing the anxiogenic effects of high dose THC. This issue needs to be resolved experimentally (see #3-4 below).\nMajor concerns:\n1) Figure S1 does not make sense. How can 80% of GABA neurons express CB1. CB1 is confined to a small subset of GABA cells in the cortex. This likely represents a technical artifact.\nRNAscope in situ hybridization (ISH) detects single mRNA transcripts with high specificity and sensitivity owing to its double-Z probe design (PMID: 22166544). Our RNAscope data aligns with MERFISH whole-brain spatial transcriptomic data from the Allen Brain Map (https://portal.brain-map.org/atlases-and-data/bkp/abc-atlas; MERFISH-C57BL6J-638850, MERSCOPE v1 whole brain), which reports that approximately ~83% of ACC GABAergic neurons express Cnr1 mRNA above a threshold of 2.5 counts per million (CPM). This independent validation argues against a technical artifact. Also, in response to Reviewer 2, we have relocated Cnr1 mRNA data from Supplementary Fig. 1 into Fig. 1g,h.\nCnr1 expression in GABA neurons\nCnr1 expression in ACC GABA neurons GABA neurons in ACC\nDistribution of Cnr1 expression in ACC GABA neurons\nFigure 1. Cnr1 mRNA is widely expressed in GABA neurons in a coronal section of mouse brain (top). Isolation of Cnr1 expression in ACC GABA neurons (middle) and distribution of expression level (bottom).\n2) Figure 2 should be supplemental as it is just tool validation and does not really add much to the main paper, and replicates other work showing sensor increases to CB1 direct and indirect agonists.\nWe agree with the reviewer and have moved the original Fig. 2 to the Supplementary Fig. 4.\n3) A major conclusion of the work is that CB receptors constrain an anxiolytic ACC-DMS circuit, and these receptors are required for THC aversion. For the latter conclusions to be fully supported, the authors need to show that activation of the ACC-DMS pathway produces place preference (using RTPP assay) and 2) that CB deletion increases glutamate release at AAC-DMS synapses or the in vivo activity of this pathway during place preference expression. Ideally (although understandably harder), CB1 deletion from this pathway should turn subthreshold optogenetic place preference into suprathreshold place preference. Without these data, there is no link between the EPM/open field data (behavior and sensor) and THC aversion data”.\nWe thank the reviewer for raising this important point and agree that establishing a mechanistic link between anxiety-related behavior, ACC→DMS circuit activity, and THC-mediated aversion is critical. To address this, we showed that optogenetic stimulation (20 Hz) of ACC axon terminals in the DMS induced real-time place preference (RTPP) in ACC→DMSCB1cKO mice, but not ACC→DMSWT mice. These results indicate that CB1 signaling constrains a positive-valence affective state induced by activation of the ACC→DMS pathway. In parallel, and in response to Reviewer 3’s comment, we examined the effects of ACC→DMS activation on anxiety-like behavior using the same stimulation parameters. Optogenetic activation of the ACC→DMS pathway produced anxiolytic-like effects in the elevated plus maze in ACC→DMSCB1cKO mice, but not in ACC→DMSWT mice. Together, these results demonstrate that place preference and anxiolysis are similarly unmasked by pathway-specific CB1 deletion, suggesting that this pathway acts as a common substrate for these behaviors.\nAlthough we did not directly measure CB1 deletion-induced increase in glutamate release or in vivo ACC→DMS activity during place preference expression, our in vitro optogenetic electrophysiological studies demonstrate robust CB1-dependent depression at ACC→DMS synapses (Fig. 1e,f), providing a functional mechanism by which CB1 deletion would be expected to enhance corticostriatal output. In addition, we find that pathway-specific CB1 deletion modifies the behavior produced by optogenetic activation of the ACC→DMS pathway (Fig. 5d-f). Finally, we show that GRAB signals in this pathway are similarly elevated by eCB2.0 anxiogenic and aversive stimuli (e.g., foot shock, Fig. 3a,b; predator odor, Fig. f-h; and open spaces, Fig. 2), supporting engagement of ACC→DMS eCB signaling during aversive states.\nTaken together, these findings support a model in which CB1 receptors constrain an anxiolytic, glutamatergic ACC→DMS circuit, and that removal of this constraint via CB1 deletion reduces THC-induced place aversion. We have revised the Discussion to clarify this mechanism.\n4) It is possible that ACC-DMS will not produce place preference alone. This would suggest that reduced THC-induced anxiety underlies the reduced place aversion. Showing that AAC-DMS CB1 deletion blocks increased anxiety induced by high dose THC would be needed to support this alternative hypothesis. Doing these studies first might preclude the necessity for #3 if this deletion clearly blocks the anxiogenic effects of high dose THC, in which case the conclusions would be that blocking anxiogenic effects of THC results in secondary reduced THC aversion.\nWe agree that THC-induced anxiety and aversion can be tightly related. Determining whether ACC→DMSCB1cKO blocks THC-induced anxiety would certainly be informative. However, two major interpretational limitations led us to prioritize other approaches in the current study. First, ACC→DMSCB1cKO mice exhibit a baseline anxiolytic-like phenotype, which would make it difficult to distinguish a genuine blockade of THC-induced anxiety from a floor effect due to their already reduced anxiety. Second, high-dose THC markedly suppresses locomotor activity. Because standard anxiety assays such as the elevated plus maze and open field depend on exploratory movement, behavioral changes observed under acute THC exposure are difficult to interpret as anxiety-specific rather than as consequences of nonspecific locomotor suppression.\n5) Changes in sensor signal after marble burying are not convincing, just as many bouts of increased activity are seen on the dot-plot. i.e. the magnitude is very small and biological significance is questionable.\nWe agree that the observed changes in GRAB signal during marble burying were very eCB2.0 small and it is not easy to interpret their functional significance. Accordingly, we have removed the fiber photometry marble burying experiments from the Results section.\nMinor concerns:\n1) The AUC data for cannabinoid sensor is hard to follow. May be best to label the baseline, pre-exit, post exit time periods on the time course data so the readers know where the AUC measures are being taken.\nWe have followed the reviewer’s suggestion and added labels for baseline, pre-exit, post exit 1, and post exit 2 time periods in the figure (revised Fig. 2).\n2) The negative fear conditioning data could also be a supplement.\nThe fear conditioning and extinction data has been moved to Supplementary Fig. 8.\n3) There is no functional dissociation of ACC to D1/D2 pathways, this at least needs to be noted as a limitation of the work. It should also be placed in context of cannabinoid plasticity segregation/specificity at cortical to D1 and D2 synapses (i.e. work of Lovinger, Malenka and others). Also ref 30 indicates asymmetric innervation of D1 over D2 neurons in the DMS.\nWe agree with the reviewer that functional dissociation of ACC inputs to D1- and D2-expressing MSNs is an important consideration. We did not directly address the distinct functional roles of D1 and D2 pathways targeted by the ACC. We have revised the Discussion to explicitly note the absence of cell type-specific analysis as a limitation of the present study. In addition, we now place our findings in the context of prior work demonstrating segregation and specificity of cannabinoid-mediated plasticity at cortical synapses onto D1 and D2 MSNs, including studies by Lovinger, Malenka, and others (e.g., PMID: 29458998).\n4) Number of biological replicates is not shown for Figure 3. The number of transitions is used for the “n” for statistical analysis but without knowing how many mice this is derived from the rigor is hard to evaluate.\nWe clarified the number of mice used in the original Fig. 3 (now Fig. 2).\n5) Do AAC-DMS neurons collateralize to innervate other targets? This might change the interpretation of the circuit-specific KO data.\nWe thank the reviewer for raising this important point. Cortical neurons that project to the striatum can indeed collateralize to innervate additional brain regions (e.g., PMIDs: 31668485, 33243980, 8922332), raising the possibility that ACC→DMS neurons targeted in our study may influence other downstream targets (acknowledged in our Discussion). While current viral and genetic strategies do not permit selective gene deletion restricted exclusively to a single axonal projection without affecting collaterals, this is a limitation of pathway-specific manipulations.\nReviewer #2 (Remarks to the Author):\nThis study investigated the role of CB1 receptor signaling in the anterior cingulate cortex (ACC) to dorsomedial striatum (DMS) pathway in regulating anxiety-like behavior and aversive responses to cannabinoids. Key findings include that CB1 receptors and enzymes involved in 2-AG synthesis and degradation are detected in the ACC and DMS. Optogenetic electrophysiology demonstrates that cannabinoids suppress excitatory transmission from ACC to DMS neurons. In vivo fiber photometry reveals increased endocannabinoid release in the DMS following administration of WIN55,212-2 or exposure to risk conditions (open arm of EPM, center of open field). Genetic deletion of CB1 in ACC→DMS neurons reduces innate anxiety-like behaviors and prevents THC-induced conditioned place aversion. The authors concluded that CB1 receptor activation in this circuit contributes to the anxiogenic effects of cannabinoids, providing insights into cannabis-induced aversive effects. The study integrates advanced methodologies, including in vivo fiber photometry, optogenetic electrophysiology, and behavioral assays, to present a well-supported conclusion. These results offer novel insights into how endocannabinoid signaling in the ACC→DMS pathway influences anxiety and aversion, addressing a significant gap in cannabinoid research.\nMajor concerns:\n1) The title should better reflect the key findings regarding the role of ACC→DMS CB1 receptor signaling in anxiety-like behavior.\nWe have renamed the title of the manuscript “CB1 receptor signaling at the cingulate-striatal circuit is anxiogenic”.\n2) The introduction should include a more comprehensive background review on the dose-dependent effects of cannabinoids, highlighting their anxiolytic and rewarding effects at low doses and anxiogenic and aversive effects at high doses. Additionally, the involvement of other brain regions such as amygdala, BNST, and NAc should be cited.\nWe have revised the Introduction to include a more comprehensive discussion of the dose-dependent effects of cannabinoids, highlighting their anxiolytic and rewarding effects at low doses and their anxiogenic and aversive effects at higher doses. In addition, we now cite and discuss prior work implicating other brain regions, including the amygdala, bed nucleus of the stria terminalis (BNST), and nucleus accumbens, in mediating these effects.\n3) ACC glutamatergic neurons likely project to multiple brain regions, a supplementary figure showing these projections would clarify the significance of the ACC→DMS pathway in this study.\nWe injected AAV9-CaMKII-ChR2-mCherry into the ACC. Brain-wide imaging shows that ACC excitatory neurons project to the DMS, thalamus, red nucleus, zona incerta and periaqueductal grey, among other regions. The DMS received a high level of ACC innervation. These results were added to the Supplementary Fig. 1.\n4) The images showing Dagla expression in D1- and D2-MSNs (Fig. 1g) and Mgll expression in ACC GABAergic and glutamatergic neurons (Extended data Fig. 2) are unclear. Higher-magnification images and more detailed methods for identifying these neurons would improve the clarity of these results. If possible, quantifying the percentage of ACC or DMS neurons expressing Dagla or Mgll would strengthen the conclusions.\nWe have now provided high-resolution images of all RNAscope experiments, including Dagla mRNA expression in D1- and D2-MSNs in the striatum (revised Supplementary Fig. 2), as well as Mgll expression in ACC glutamatergic (Slc17a7 for vGluT1) and GABAergic (Slc32a1 for vGAT) neurons (revised Supplementary Fig. 3). For quantification, we reconstructed a surface for cell-specific markers (Drd1 vs. Drd2; Slc17a7 vs. Slc32a1) using Imaris and applied the Spots function to identify Dagla/Mgll puncta with a threshold of 1 μm diameter. Our analysis revealed that Dagla mRNA was ubiquitously expressed (100%) in D1- and D2-MSNs in the striatum while Mgll mRNA was also ubiquitously expressed in excitatory (vGluT1+) and GABAergic (vGAT+) neurons in the ACC. We have clarified this in the revised manuscript. As a result of ubiquitous expression of these mRNAs, we only presented puncta density in each cell types.\n5) According to the retrograde endocannabinoid signaling hypothesis, CB1 activation reduces eCB (2-AG) release (Fig. 2g), yet WIN55,212-2 induces a robust increase in eCB signaling via a CB1-dependent mechanism (Fig. 2c). This apparent contradiction should be addressed.\nWIN55212-2 was used in the eCB signal assays, while THC was used in the CPA experiments. It would be valuable to determine whether THC similarly increases eCB release in the DMS as WIN55212-2 does.\nWe appreciate the reviewer’s thoughtful question and the opportunity to clarify this apparent discrepancy. We agree that activation of presynaptic CB1 receptors by endogenous 2-AG or exogenous agonists can suppress neurotransmitter release and thereby secondarily reduce activity-dependent eCB production. In our study, the experiment using WIN55,212-2 was performed as a pharmacological validation of GRAB sensor, rather than as a test of retrograde eCB regulation. Although GRAB is a modified CB1 receptor optimized to report eCB2.0 eCBs (primarily 2-AG), it can also be directly activated by synthetic CB1 agonists. In the original characterization of GRAB , WIN55,212-2 was shown to exhibit near-maximal efficacy eCB2.0 (~100% relative to 2-AG) at the sensor (PMID: 34764491). Accordingly, the robust fluorescence increase induced by WIN55,212-2 reflects direct activation of the sensor rather than increased eCB release. The reviewer raises the valuable point of whether THC similarly alters eCB signaling in the DMS. However, THC exhibits very low efficacy at GRAB (Emax ~10% of 2- eCB2.0 AG) (PMID: 34764491). For this reason, we did not pursue THC in our fiber photometry assays.\n6) The authors claim that eCB signaling increases as mice exit closed arms and enter open arms of the EPM and decreases in the opposite transition. However, data in Fig. 3 (d, f, e, g) suggest that GRABeCB2.0 signals similarly decline after both closed and open arm exits or entry. Further clarification and discussion of these findings are needed.\nWe observed that GRAB fluorescence typically began to rise prior to the actual exit from eCB2.0 the closed arm and peaked in the center area. Because mice approach the center cautiously, this process usually lasted ~10 seconds. To capture this dynamic, we analyzed longer time windows before and after the transitions to establish a stable baseline. Once in the center or open arms, mice generally remained only briefly before returning to a closed arm, at which point GRAB fluorescence declined. Thus, most events followed a stereotyped trajectory: starting eCB2.0 in a closed arm, passing through the center/open arm, and returning to another closed arm. This explains why closed-arm exits and entries appear similar in the averaged traces, since in many cases they were separated by only 1–2 seconds. Consistently, GRAB signal decreased eCB2.0 shortly after closed-arm re-entry. In contrast, entries into open arms were often followed by longer dwell times, during which GRAB fluorescence remained elevated for a slightly eCB2.0 extended period (revised Fig. 2h). Exiting the open arms was associated with a rapid decline in signal. Because the duration and dynamics of individual events varied substantially, Z-scoring across multiple events would have been impossible. In response to Reviewer 2, we highlight the temporal changes in GRAB fluorescence around EPM events by denoting 5 second eCB2.0 intervals for Baseline, Pre-entry, post-entry 1, and post-entry 2 with unique colors (see Fig. 2). We also provided spatial maps of GRAB signal as an independent way of illustrating these eCB2.0 dynamics. Importantly, the sustained elevation of GRAB signal during open-arm exploration eCB2.0 may reflect prolonged engagement of eCB/CB1R signaling in anxiogenic contexts, consistent with a role for this pathway in modulating avoidance and coping behaviors.\n7) While Extended Data Fig. 5 provides electrophysiological evidence of CB1 deletion, additional IHC or RNAscope images confirming CB1 loss specifically in ACC→DMS neurons would be beneficial. If unavailable, Extended Data Fig. 5 should be relocated to the main text.\nWe appreciate the reviewer’s suggestion to further validate CB1 deletion using anatomical approaches. However, there are important technical limitations that prevent IHC or RNAscope from providing unambiguous, projection-specific confirmation of CB1 loss in ACC→DMS neurons. CB1 receptors are broadly and densely distributed across corticostriatal circuits and are predominantly localized to presynaptic axon terminals (e.g., PMID: 29466446), making it difficult to distinguish pathway-specific deletion using IHC alone. In our experience, combining IHC with RNAscope greatly reduces the strength of IHC signals. In contrast, electrophysiological recordings provide a direct functional readout of CB1-mediated synaptic modulation at ACC→DMS synapses, thereby offering rigorous validation of effective CB1 deletion in this pathway. We have followed the reviewer’s recommendation and relocated Extended Data Fig. 5 to revised Fig. 4a,b to emphasize this key validation experiment.\n8) The behavioral data in Fig. 4e and Fig. 4k appear inconsistent and should be clarified.\nThe apparent discrepancy arose from typographical errors in the figure legends, which have now been corrected. Specifically, “Center time” in the original Fig. 4e refers to the open field test (revised Fig. 4g), whereas “Center time” in the original Fig. 4k refers to time spent in the center square of the elevated plus maze (revised Fig. 4m).\n9) 25 mg/kg THC dose used in the CPA experiment is quite high and could induce sedation, hypothermia, or motor impairments, potentially confounding place preference results. A lower dose should be tested, or at minimum, these concerns should be addressed in the discussion.\nWe agree that acute behavioral effects of high doses of THC can confound behavioral assays that depend on locomotion, such as open field or elevated plus maze tests. However, in the CPA paradigm used here, mice were confined to a single chamber during THC or vehicle pairing, and the final preference test was conducted in a drug-free state. This design minimizes potential confounds arising from THC-induced locomotor suppression. Studies using lower THC doses show mixed results (for review, see PMID: 20495676). We selected 25 mg/kg because it produces a robust and consistent CPA, allowing us to draw clear conclusions from our experiments.\n10) The study attributes THC-induced conditioned place aversion solely to CB1 receptor activation in the ACC→DMS pathway. However, systemic THC effects on other circuits should be acknowledged in the discussion.\nWe have revised the Discussion to clarify that our findings do not imply that THC-induced CPA arises exclusively from this circuit but rather identify ACC→DMS CB1 signaling as a critical contributing component within a broader network of THC-responsive pathways.\n11) The hypothesis that \"inhibition of ACC terminals in the DMS causes anxiety-like behavior and thereby contributes to the aversive effects of THC\" does not account for how low doses of cannabinoids or THC produce anxiolytic effects. The potential involvement of other CB receptors, neuronal subpopulations, or circuits in cannabinoid-induced reward and anxiolysis should be discussed.\nWe have improved our discussion to better incorporate our findings with anxiolytic effects of cannabinoids at other loci in the brain.\nMinor Points\n1. THC is not a selective CB1 receptor agonist (line 56). This should be revised for accuracy.\nIndeed, THC is a non-selective partial CB1 agonist, and our previous statement has been revised for accuracy.\n2. WIN55,212-2, at 5 mg/kg, robustly increases eCB signaling in the DMS. Is this effect dose-dependent? Additional clarification would be useful.\nWIN55,212-2 was used to validate the GRAB sensor rather than to characterize dose– eCB2.0 response effects on eCB signaling. Accordingly, dose dependence was not examined in the present study.\n3. In electrophysiology and RNAscope experiments, the sample sizes refer to the number of neurons or brain sections. State the number of mice used in these experiments.\nWe have revised all relevant figure legends to include the number of mice used in each experiment.\n4. Figure Relocation Suggestion: Move Fig. 1g and Fig. 5 (a, b, c) to the supplementary materials while relocating Extended Data Figs. 1, 4 and 5 to the main figures for better organization.\nWe have reorganized the figures by following the reviewer’s suggestions.\nReviewer #3 (Remarks to the Author):\nReviewer comments on Manuscript NCOMMS-25-06847-T \"CB1 receptor signaling at the cingulate-striatal circuit is aversive\"\nThis manuscript aims to understand neural mechanisms for high dose THC effects in the brain through several experiments done focusing on the dorsal medial striatum (DMS) inputs from the anterior cingulate (ACC). They use a range of behavioral experiments, slice electrophysiology, and in situ hybridization to find the role of cannabinoid receptors in mostly aversive effects of THC. Authors use optogenetics and electrophysiology to show the monosynaptic connection between ACC and DMS and the presence of CB1Rs from the ACC terminals at the DMS. Moreover, with the presence of the synthesis enzyme for endocannabinoid 2-AG (DAGLa) in the DMS (with no preference to D1 or D2 medium spiny neurons), authors showed 2-AG mediated short-term depression of glutamatergic transmission (DSE). They continued in vivo physiology recordings by using the GRABeCB2.0 sensor and showed the endocannabinoid binding increases on those terminals in freely moving mice in elevated plus maze and open field tests while mice cross through the open arms (EPM) or the center of the brightly lid arena (OF) which are mostly anxiogenic for mice (innately) and often avoided. Recordings on the same terminals during fear conditioning and extinction learning only showed increased binding during shock onset but not during tone onset/offset. Using an intersectional approach, they knocked down CB1Rs on those terminals and showed mice became less anxious and avoid the high dose THC paired compartment on the conditioned place preference test.\nThis manuscript is written well in a clear language explaining the reasoning of the experiments done and discuss the results obtained without speculation. Please find questions and comments to improve the manuscript.\n1- Authors showed the involvement of the CB1Rs in the ACC terminals at DMS in avoidance of aversive/stressful events. DMS majorly plays role in goal directed behaviors and have low expression of CB1Rs. Could the low expressed CB1Rs in DMS (compared to DLS) be involved in important roles in survival behavior? Maybe authors should try the fear conditioning with a predator odor to test the idea of increased endocannabinoid signaling in innate but not learned aversive stimuli. Testing their intersectional knockout mice on the predator odor conditioning and recording the terminals during the predator odor conditioning test will solidify their findings and their proposed model and neural mechanism.\nWe thank the reviewer for this insightful comment. The reviewer is correct in pointing out that the CB1 receptor expression is lower in the DMS compared to DLS (PMID: 29466446). Indeed, an intriguing possibility is that CB1 receptors at ACC→DMS synapses are preferentially activated during conditions associated with heightened eCB release, such as risky or threatening situations, thereby contributing to survival-relevant behavioral regulation. In response to the reviewer’s question, we performed new experiments examining whether the predator odor 2-methyl-2-thiazoline (2-MT) induces eCB release at the ACC→DMS pathway. We found that 2-MT induced a rapid and robust increase in GRAB signal on ACC terminals eCB2.0 in the DMS (revised Fig. 3f-h). Together with our findings that GRAB signals are similarly eCB2.0 elevated by other anxiogenic or aversive stimuli (e.g., foot shock and open spaces), these results indicate that ACC→DMS eCB signaling is strongly engaged during innate threat and stress states, but not by conditioned cues alone. While testing ACC→DMSCB1cKO on predator odor conditioning would indeed provide valuable insight, such experiments would require substantial additional resources and are best addressed in future studies.\n2- Conditioned place aversion test for a high dose THC applied on multiple conditioning days before the test day brings the question on how the mice behave on 6th day. Have they determined this with tetrad tests? How is the time spent in vehicle side in this experiment? Please plot and show side by side. t-test may not be the best to test the genotype +test phase related changes, please use 2-way ANOVA.\nWe did not perform a full cannabinoid tetrad battery on the test day. However, mice did not receive THC on the test day, so the final CPA test was conducted in a drug-free state. Thus, acute effects of THC cannot account for the observed place aversion. Nonetheless, locomotor activity during the testing day was moderately reduced compared to the pretest day, which could be attributed to acclimation to the chamber (Supplementary Fig. 10). In response to the reviewer’s request, we now explicitly plot the time spent in both the THC-paired and vehicle-paired chambers side by side (Fig. 6c,d). These data show that reduced occupancy of the THC-paired side is accompanied by a corresponding increase in time spent in the vehicle-paired side, consistent with a true shift in place preference rather than generalized behavioral suppression.\nWe agree that a t-test is not optimal for evaluating genotype-dependent changes across test phases. Accordingly, we have reanalyzed the CPA data using a two-way ANOVA with genotype and test phase as factors, followed by appropriate post hoc comparisons. This analysis confirms the original conclusions, and the statistical approach has been updated throughout the manuscript.\n3- Have the authors manipulated this circuit during behavior? Activation/inhibition of ACC-DMS projections in the open field and EPM tests (or now suggested predator odor conditioning test).\nIn response to the reviewer’s comment, we conducted new experiments examining optogenetic excitation of the ACC→DMS pathway in the elevated plus maze and open field tests in ACC→DMSWT and ACC→DMSCB1cKO mice. We found that optogenetic activation led to an increase in the time spent in the open arms of the elevated plus maze in ACC→DMSCB1cKO mice compared to ACC→DMSWT mice but had no effect on time spent in the center zone of the open field (please see also our response to Reviewer 1, Question 3). These results have been summarized in newly added Fig. 5g-j and Supplementary Fig. 9.\n4- Authors don’t show any data regarding the influence of the GABAergic interneurons in DMS that may be important in modulating avoidance of stressful/aversive stimuli. Specifically, D1R expressing MSN’s and the possible involvement of anandamide should be tested in slice electrophysiology experiments.\nWhile our present study does not directly examine the influence of GABAergic interneurons in the DMS or test the specific involvement of D1R MSNs and anandamide through slice electrophysiology, we agree with the reviewer that these are critical aspects warranting future investigation. Notably, recent work provides compelling evidence that eCB signaling, particularly via anandamide and CB1 receptor activation, plays a pivotal role in regulating stress-induced plasticity and excitatory-inhibitory balance in somatostatin-expressing (SOM+) interneurons in the nucleus accumbens (PMID: 37590414). Their study demonstrates that acute stress increases the intrinsic excitability of SOM+ interneurons and reorganizes glutamatergic inputs, an effect that is fully dependent on eCB signaling at CB1 receptors. We have added a brief discussion in the revised manuscript.\n5- Please be more specific in baseline periods for fiber photometry experiments.\nWe have improved the clarity of the fiber photometry figures and legends.\n6- Please include a small discussion on the role of ACC-DMS neurons in itching /scratching (https://doi.org/10.1038/s41467-020-16859-5) which resembles to the reaction to excessive stress.\nWe thank the reviewer for this valuable suggestion. The ACC is an important cortical region for top-down modulation of itch sensation (PMIDs: 32546780, 41131169, 29540548). Similar to threat exposure, itch involves detection of a potentially harmful signal that elicits rapid behavioral responses such as scratching. We have added a brief discussion highlighting the relevance of ACC control of other aversive stimuli, including itch, to further contextualize our findings.\nOther issues related to the figures and their corresponding legends:\nFigure 1: Delete repeated words on legend line 558 ‘depolarization induced’.\nWe have corrected the error identified by the reviewer.\nFigure 2: Move the pharmacological validation figure into supplementary figures. Instead show one of the experiments suggested above.\nWe have followed the suggestion of this reviewer, as well as Reviewer 1, and relocated the pharmacological validation figure to Supplementary Fig. 4.\nFigure 3: Does the line in time 0 indicate the event start? (ie. crossing to the other compartment) Explain why the baseline is -20 to -15 s in figure 3d. Shouldn’t it be 5 s before the entry? Indicate the times for baseline, pre-entry, post entry 1 and post entry 2 into the figure legends and also to the methods.\nThe time 0 indicates crossing from one compartment to the other. Fiber photometry recordings in EPM transitions compare 5 s prior to (Pre-event) and after (Post-event 1) compartment changes in addition to other periods via a one-way RM ANOVA. Please see our response to Reviewer 2 (Question 6) where we addressed the rationale for our chosen time course, baseline, and post event periods. In short, mice approached the center of the EPM slowly, which was associated with a gradual increase in GRAB fluorescence. We show a longer eCB2.0 time course before and after each event (20 s), so that this can be appreciated. A baseline of - 20 to -15 s was chosen since most mice were far away from the center or open arms at this point. We have incorporated the specific details for the timing of GRAB signal in the EPM eCB2.0 (Baseline: -20 to -15 s, Pre-event: -5 to 0 s; Post-event 1: 0 to 5 s; Post-event 2: 5 to 10 s) in the figure, legends and methods. In response to the reviewer, we have clarified the legend for Fig. 2 and added this information into the Methods section.\nFigure 4: The legends and the figures shown are not consistent thus confusing to the reader, please correct the letters and delete all words (such as: left, right, center) to make it clear by assigning each figure a letter. Please make new graphs to indicate the total distance moved in each compartment (open, closed arms and center) for EPM. CB1R ko mice are not moving much in the open field (as reflected by total distance travelled difference in ko mice) therefore could they be staying more in the center of the open field? Same problem in the EPM time in center graph doesn’t match the heat map image. Include the velocity of the mice in each compartment of the EPM test.\nAll figure panel labels and legends have been corrected to ensure consistency, and directional descriptors (e.g., left, right, center) have been removed. Each panel is now clearly identified by a letter and corresponds unambiguously to the legend.\nIn response to the Reviewer’s comment, we have added new graphs quantifying the distance traveled in each compartment of EPM (Supplementary Fig. 6). Additionally, we added Supplementary Fig. 5 to show distance traveled and velocity in the outer and center zones of the open field. Although ACC→DMSCB1cKO mice exhibited reduced total distance traveled during the open field test, their initial locomotor activity and maximal speed were not significant different from controls (Fig. 4h,i). The subsequent reduction in locomotion is therefore unlikely to reflect motor impairment and is more consistent with faster habituation to the novel environment, which is another indication of anxiolytic-like phenotype, along with the increased center time in the open field (Fig. 4f,g) and increased open-arm exploration in the EPM (Fig. 4j,k,l). These analyses and clarifications have been incorporated into the revised manuscript.\nSame problem in the EPM time in center graph doesn’t match the heat map image. Include the velocity of the mice in each compartment of the EPM test.\nWe have corrected these errors in Fig. 4. The velocity for each compartment in the EPM has been added to Supplementary Data Fig. 6.\nFigure 5: Indicate what comparisons are done for the statistical tests. For example, in extinction result of the two-way ANOVA comparisons are from day1 to day 6. Legend ‘c’ should be changed to ‘d’. Please discuss the variation in the THC response in the CPA test.\nFor fear extinction we used a 2-way RM ANOVA to compare % time freezing during tone blocks across all 6 days. The legend for Fig. 5 (now presented as Supplementary Fig. 8 in the revision) has been fixed. Indeed, there is variation in the response to THC in the CPA test, we increased the N in response to your point.\nExtended Figure 1: be more specific, include details about the number of mice used for the RNAScope studies. And how many sections were counted from each mouse. Were they counted from 40x images?\nWe have provided the number of mice, and the number of sections used for the RNAScope studies in the legend of revised Supplementary Fig. 2 (previously Extended Data Fig. 1). The images were taken with a 25x/0.95 NA/2.4 mm WD HC FLUOTAR water immersion lens and analyzed with Imaris. The information has been added in the Methods section.\nExtended Figure 2: correct ‘Cnr1’ to ‘Mgll’\nThank you for catching this, we have corrected this error.\nWe thank the three reviewers for their time and effort which have greatly improved our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have appropriately addressed my concerns. The new optogenetic data helps solidify the underlying function of the ACC-DMS pathway under examination. The data presentation has been made clearer.", "reviewer_2": "The authors have addressed all of my comments appropriately. I have no additional comments.", "reviewer_3": "Authors answered most of the questions and added new suggested experiments by the reviewers. Manuscript reads well and improved significantly."}, "author_response": ""}]

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