The interconnected wirings between medial prefrontal cortex and claustrum and their governing roles in the methamphetamine reward memory in male mice

Domain: neuroscience
Published: 2026-01-12
Editorial outcome: major_revision

Abstract
Bidirectional communication between medial prefrontal cortex (mPFC) and claustrum (CL) plays a critical role in drug reward memory, particularly for conditioned drug-associated cues. However, their precise circuitry remains unclear. Here, using viral tracing, chemo- & opto-genetics and patch-clamp, alongside methamphetamine (METH) conditioned place preference (CPP) model, we dissected the connectivity pattern between mPFC and CL in male mice. Two relatively independent pathways of top-down mPFC-CL and bottom-up CL-mPFC were identified, each comprising both direct excitatory and indirect inhibitory projections. The mPFC-CL pathway predominantly mediates inter-hemispheric communication, while the CL-mPFC pathway mainly supports intra-hemispheric connections. Under physiological conditions, strong feedforward inhibition (FFI) via local GABAergic interneurons suppresses excitatory transmission between mPFC and CL. During METH CPP, FFI is weakened, resulting in enhanced excitatory signaling. The mPFC-CL pathway was engaged during reward memory encoding, whereas the CL-mPFC pathway mediated memory retrieval. These findings suggest potential therapeutic interventions for METH reward memory.

Introduction
The brain operates as a highly interconnected wirings, which supports various cognitive and behavioral functions. Although high-tech brain science and technology have accelerated the understanding of brain networks, there are still questions that need to be clarified about structural connections, functional interactions, and efficiency connections in brain networks. As a highly addictive psychostimulant, methamphetamine (METH) abuse remains a global crisis1. The addictive potential of METH arises from its ability to profoundly influence the reward circuits in the brain, leading to euphoric and reinforcing effects2,3. Chronic METH use is tightly linked to specific environmental settings, forming strong contextual associations with METH rewards4,5.

The conditioned place preference (CPP) is a widely used behavioral paradigm to assess drug reward learning and environmental cue associations6. The CPP procedure contains two main phases: the training phase establishes (encodes) the association of the environment cue with the drug’s rewarding effects, while the test phase measures the retrieval of drug-context reward memory7.

Recently, the interconnectivity of medial prefrontal cortex (mPFC) with the claustrum (CL) has gained much attention in substance use disorders, due to its crucial role in reward-based learning and memory8,9. The mPFC is a high-level executive region of the brain, being essential for decision-making, cognitive flexibility, impulsive control, reward encoding and motivation10. The mPFC is composed primarily of glutamatergic (Glu) neurons (mPFCGlu) and a small proportion of gamma-aminobutyric acid (GABA) interneurons (mPFCGABA)11. These mPFCGABA neurons are crucial for maintaining the excitatory/inhibitory (E/I) balance of mPFCGlu neurons12,13. Hu et al. proposed that maintaining the E/I balance in the mPFC during the process of memory retrieval might represent an effective therapeutic strategy for the treatment of METH addiction14. The CL is a thin sheet-like subcortical brain structure buried between insula and putamen, densely connecting the cortical regions with other subcortical regions, serving as a hub for its role in multisensory integration, attention and consciousness15–17. Similar to the mPFC, the CL consists of approximately 90% CLGlu neurons and about 10% CLGABA neurons18. Recent emerging evidence, including ours, demonstrates that the CL plays an important role in drug-craving and seeking behaviors as well as withdrawal symptoms, which might be driven by its role in integrating environmental cue information in the drug reward process9,19,20. There exist abundant projection connections between mPFC and CL, with Glu neurons being dominant21, forming the top-down mPFC-CL pathway and the bottom-up CL-mPFC pathway. Recently, several research groups have begun to identify the interconnection patterns between mPFC and CL, as well as their role in the reward process and neurological disorders. For example, the CL provides strong and long-lasting feedforward inhibition (FFI) of the prefrontal cortex (PFC), and plays a unique role in regulating cognitive processes in prefrontal circuits22. In addition, optogenetic activation of the CL-prelimbic cortex (PL) pathway suppresses the chronic social defeat stress-induced depressive-like behaviors in male mice23. These studies suggest that the reciprocal projections between mPFC and CL involves in the process of drug reward memory, particularly for conditioned drug-associated cues. However, their precise neural circuits and the principles established therein remain unclear, leading to poorly understanding of their roles in reward.

In the current study, using viral tracing, fiber photometry, chemo- & opto-genetics and patch-clamp, alongside METH CPP model, we dissected the connectivity pattern between mPFC and CL from the view of whole brain, including the direct and indirect pathways, inter-hemispheric and intra-hemispheric communication, the overlapped receiving and projecting Glu neurons in each local region, and the physiological intermodulation between the two regions. Further, we explored the distinct governing roles of the top-down mPFC-CL and the bottom-up CL-mPFC pathways in the encoding and retrieval processes of the METH reward-associated environment context.

Results
Reciprocal projections between mPFC and CL
To validate the reciprocal connections between mPFC and CL from a whole-brain perspective, we performed a fluorescent micro-optical sectioning tomography (fMOST) experiment. As shown in Fig. 1a, b and Supplementary Fig. 1a, b, mPFC fibers chiefly projected to the contralateral CL, while CL fibers primarily targeted the ipsilateral mPFC. For the top-down mPFC-CL pathway, as shown in Supplementary Fig. 1c, d, anterograde labeling from mPFC showed EGFP-positive axon terminals were predominantly localized in contralateral CL. As shown in Supplementary Fig. 1e, f, retrograde labeling from CL revealed mCherry-positive somas were mainly in contralateral mPFC. For the bottom-up CL-mPFC pathway, as shown in Supplementary Fig. 1g, h, anterograde labeling from CL showed EGFP-positive axon terminals were predominantly in the ipsilateral mPFC. As shown in Supplementary Fig. 1i, j, retrograde labeling from mPFC revealed mCherry-positive somas were mainly observed in ipsilateral CL.

Independent features in interconnectivity between mPFC and CL
First, we identified the neuronal populations that send efferents and receive afferents within the mPFC-CL circuits (Fig. 1c–j). As shown in Fig. 1c, d and Supplementary Fig. 1k, l, projection-specific viral tracing revealed two distinct populations of mPFC neurons, as indicated by mCherry-positive neurons projecting to CL (top-down pathway) and EGFP-positive neurons receiving inputs from CL (bottom-up pathway). Minimal overlap between these two populations, which indicates that the projecting neurons and the receiving neurons within the mPFC are distinct populations. As shown in Fig. 1e, f, it was about 73.5% of mPFCGlu neurons while 17.4% mPFCGABA neurons that received afferents from CL, implying CL-mPFCGlu pathway and CL-mPFCGABA pathway. By contrast, about 92.3% of neurons that send efferents to CL were mPFCGlu neurons. A similar investigation was carried out to identify CL neurons (Fig. 1g, h and Supplementary Fig. 1m, n), as indicated by mCherry-positive neurons projecting to mPFC and EGFP-positive neurons receiving inputs from mPFC. Minimal overlap between these two populations, which indicates that the projecting neurons and the receiving neurons within the CL are distinct populations. As shown in Fig. 1i, it was about 67.3% of CLGlu neurons while 24.9% CLGABA neurons that received afferents from mPFC, implying mPFC-CLGlu pathway and mPFC-CLGABA pathway. By contrast, about 91.8% of neurons that send efferents to mPFC were CLGlu neurons (Fig. 1j). Together, these results indicate that projecting neurons and input-receiving neurons are largely distinct within both mPFC and CL. These projecting neurons are mainly Glu, while the receiving population consisted of a mixture of Glu and GABA neurons.

Second, we used electrophysiological patch-clamp recording to investigate the potential microcircuit architecture and functional connectivity between the mPFC and CL (Figs. 2 and 3). As shown in Fig. 2a and Supplementary Fig. 2a, b, the EGFP-labeled CLGABA neurons, while dense mCherry-labeled mPFCGlu terminals within CL. As shown in Fig. 2b, c, both excitatory postsynaptic currents (oEPSCs) and inhibitory postsynaptic currents (oIPSCs) were induced on CLGlu neurons when opto-stimulating mPFCGlu terminals. Both oEPSCs and oIPSCs of CLGlu neurons were abolished by incubating tetrodotoxin (TTX) in the ACSF, and the oEPSCs but not oIPSCs were reintroduced by the application of 4-aminopyridine (4-AP). As shown in Fig. 2d, e, bath application of picrotoxin (PTX) blocked the oIPSCs but not oEPSCs, while bath application of 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(f) quinoxaline (NBQX) and D-2-Amino-5-phosphonovaleric acid (AP5) blocked both oEPSCs and oIPSCs of CLGlu neurons. These results suggest that mPFCGlu neurons make monosynaptic excitatory connections and disynaptic inhibitory connections with CLGlu neurons. When opto-stimulating mPFCGlu terminals, the oEPSCs were induced on CLGABA neurons, which were abolished by TTX and then reintroduced by 4-AP and abolished again by bath application of NBQX and AP5 (Fig. 2f–h). These results suggest that mPFCGlu neurons make monosynaptic excitatory connections with CLGABA neurons. Then, we verified the monosynaptic connections from CLGABA neurons to CLGlu neurons. As shown in Fig. 2i and Supplementary Fig. 2c, d, the EYFP-labeled CLGABA neurons that received mPFCGlu terminals. When opto-stimulating CLGABA neurons, the oIPSCs were induced on CLGlu neurons, which were abolished by TTX, reintroduced by 4-AP and then abolished by PTX again (Fig. 2j, k). These results suggest that CLGABA neurons make monosynaptic inhibitory connections with CLGlu neurons. Taken together, these patch-clamp results verified the existence of mPFCGlu-CLGlu direct excitatory pathway and mPFCGlu-CLGABA-CLGlu indirect inhibitory pathway.

Similarly, as shown in Fig. 3a and Supplementary Fig. 2e, f, the EGFP-labeled mPFCGABA neurons, while dense mCherry-labeled CLGlu terminals within mPFC. As shown in Fig. 3b, c, both oEPSCs and oIPSCs were induced on mPFCGlu neurons when opto-stimulating CLGlu terminals. Both oEPSCs and oIPSCs of mPFCGlu neurons were abolished by incubating TTX in the ACSF, and the oEPSCs but not oIPSCs were reintroduced by the application of 4-AP. As shown in Fig. 3d, e, bath application of PTX blocked the oIPSCs but not oEPSCs, while bath application of NBQX and AP5 blocked both oEPSCs and oIPSCs of mPFCGlu neurons. These results suggest that CLGlu neurons make monosynaptic excitatory connections and disynaptic inhibitory connections with mPFCGlu neurons. When opto-stimulating CLGlu terminals, the oEPSCs were induced on mPFCGABA neurons, which were abolished by TTX and then reintroduced by 4-AP and abolished again by bath application of NBQX and AP5 (Fig. 3f–h). These results suggest that CLGlu neurons make monosynaptic excitatory connections with mPFCGABA neurons. Then, we verified the monosynaptic connections from mPFCGABA neurons to mPFCGlu neurons. As shown in Fig. 3i and Supplementary Fig. 2g, h, the EYFP-labeled mPFCGABA neurons that received CLGlu terminals. When opto-stimulating mPFCGABA neurons, the oIPSCs were induced on mPFCGlu neurons, which were abolished by TTX, reintroduced by 4-AP and then abolished by PTX again (Fig. 3j, k). These results suggest that mPFCGABA make monosynaptic inhibitory connections with mPFCGlu neurons. Taken together, these patch-clamp results verified the existence of CLGlu-mPFCGlu direct excitatory pathway and CLGlu-mPFCGABA-mPFCGlu indirect inhibitory pathway.

Collectively, there are four independent pathways between mPFC and CL across the two cerebral hemispheres, the top-down mPFC-CL pathway which primarily facilitates communication between the left and right hemispheres of the brain, as well as the bottom-up CL-mPFC pathway that predominantly connects regions within the ipsilateral hemisphere (Fig. 3l). As shown in Fig. 3m, in either top-down pathway of mPFC-CL or bottom-up pathway of CL-mPFC, there exist both direct excitatory pathways and indirect inhibitory pathways between mPFCGlu neurons and CLGlu neurons, forming Glu-Glu pathways and Glu-GABA-Glu pathways in mPFC-CL circuits. The projecting Glu neurons are rarely overlapped with the receiving Glu neurons in each region of interconnectivity pathways between mPFC and CL, which are connected anterogradely to form an unclosed circuit.

Physiological regulation of the mPFC-CL circuits
To investigate functional connectivity between mPFC and CL in vivo, we monitored neuronal activity during pathway-specific activation (Fig. 4 and Supplementary Fig. 3). For the top-down mPFC-CL pathway (Fig. 4a, b and Supplementary Fig. 3a), activation of mPFC neurons significantly increased the activity of CLGABA neurons, while suppressing CLGlu neurons (Fig. 4c, d and Supplementary Fig. 3b, c). Similarly, for the bottom-up CL-mPFC pathway (Fig. 4e, f and Supplementary Fig. 3d), when activating the CL neurons enhanced mPFCGABA neuronal activity and reduced mPFCGlu neuronal activity (Fig. 4g, h and Supplementary Fig. 3e, f).

These results indicate that both top-down (mPFC-CL) and bottom-up (CL-mPFC) pathways exhibited a reciprocal pattern under physiological conditions, Glu neurons at one end negatively regulated Glu neuronal activity at the other end. This effect is likely mediated by strong FFI via GABAergic interneurons in the mPFC-CL circuits (Fig. 4i, j).

Activity phenotypes of mPFCGlu and CLGlu in METH CPP
To investigate the neural activity in mPFC and CL under pathological conditions, we established a METH CPP model in male mice. After 8 days of CPP-Training, the mice exhibited a significantly increased preference for the drug-paired chambers (Fig. 5a, b and Supplementary Fig. 4a, b). Following CPP-Test, the ramp-shaped action potentials (APs) were recorded in slices containing mPFC and CL. Compared with that of saline group, the number of APs in mPFC and CL neurons of METH group was significantly higher after ramp-shaped injection of cellular current (Fig. 5c–f and Supplementary Fig. 4c, d). In addition, as shown in Supplementary Fig. 4e–h, compared with saline group, both the c-Fos (a marker of neuronal activation)-positive mPFCGlu neurons and c-Fos-positive CLGlu neurons were significantly increased, while the c-Fos-positive mPFCGABA neurons and c-Fos-positive CLGABA neurons remained unchanged after CPP-Test in METH group mice.

Next, the oEPSCs and oIPSCs of mPFCGlu neurons and CLGlu neurons along the interconnectivity circuits between mPFC and CL were recorded in saline group and METH group after CPP-Test (Fig. 5j–l). As shown in Fig. 5g–i, compared with that in saline group, the oEPSCs amplitude was significantly increased, while the oIPSCs amplitude of CLGlu neurons was significantly decreased in METH group mice. As shown in Fig. 5j–l, compared with that in saline group, the oEPSCs amplitude was significantly increased, while the oIPSCs amplitude of mPFCGlu neurons was significantly decreased in METH group mice. These results indicate that the neuronal activities of mPFCGlu neurons, mPFCGABA neurons, CLGlu neurons and CLGABA neurons during METH CPP-Test have changed compared with the physiological state. As for the indirect pathways along mPFC-CL and CL-mPFC interconnectivity, it seems that the inhibitory effects of both CLGABA micro-projections on CLGlu neurons and mPFCGABA micro-projections on mPFCGlu neurons were weakened following CPP-Test in METH group mice, which leads to a synchronous increase in the activities of mPFCGlu neurons and CLGlu neurons (Fig. 5m, n).

The top-down mPFC-CL pathway contributes to the encoding of METH reward memory during the process of METH CPP-Training
To investigate the role of the mPFC-CL pathway in METH CPP, the top-down pathway was regulated during METH CPP-Training period (Group 1 and Group 3) or during METH CPP-Test period (Group 2 and Group 4).

First, we performed chemogenetic manipulation to suppress the mPFC neurons along the mPFC-CL pathway (Fig. 6a, b and Supplementary Fig. 5a, b). As shown in Supplementary Fig. 5c, d, approximately 50% transfection rate in mPFCGIu neurons and clozapine-N-oxide (CNO) can effectively reduce mPFC neuronal activity. As shown in Supplementary Fig. 5e, f, compared to the Veh-treated mice of METH group (M-Veh group), the CNO-treated mice of METH group (M-CNO group) during CPP-Training period (Group 1) effectively reduced the percentage of c-Fos-positive CLGlu neurons, but this effect was not observed during CPP-Test period (Group 2). However, the M-CNO group during either CPP-Training period (Group 1) or CPP-Test period (Group 2) had no effect on the percentage of c-Fos-positive CLGABA neurons (Supplementary Fig. 5g, h). As shown in Fig. 6c, d (Group 1), compared to the corresponding Veh group, inhibiting the mPFC neurons projecting to CL during CPP-Training period had no effect on CPP score and ΔCPP score in the CNO-treated mice of saline group (S-CNO group), but effectively reduced the CPP score and ΔCPP score in M-CNO group. There was no difference in the total distance traveled among the four groups of mice. However, as shown in Fig. 6e, f (Group 2), when inhibiting these neurons during CPP-Test period, compared to the corresponding Veh group, both S-CNO group and M-CNO group exhibited similar CPP score and ΔCPP score. There was no significant difference in the total distance traveled among the four groups of mice. Additionally, chemogenetic inhibition of mPFCGlu terminals in the CL during CPP-Training period also reduced the CPP score and ΔCPP score in M-CNO group mice (Supplementary Fig. 6a–d), and effectively suppressed the activity of Glu neurons in the CL (Supplementary Fig. 6e, f) following CNO infusion.

Then, we found that the CLGABA neurons in the mPFC-CL pathway showed significantly reduced activity during CPP-Training period in METH group mice when compared to the saline group mice, while the CLGABA neurons activity had no significant changes during CPP-Test period (Supplementary Fig. 7a–c). Specifically, activating the CLGABA neurons along the mPFC-CL pathway through chemogenetic regulatory techniques (Fig. 6g, h and Supplementary Fig. 8a, b). As shown in Supplementary Fig. 8c, d, with 60% transfection rate in CLGABA neurons and CNO can effectively enhance CL neuronal activity. As shown in Supplementary Fig. 8e, f, compared to the M-Veh group, the M-CNO group during CPP-Training period (Group 3) effectively reduced the percentage of c-Fos-positive CLGlu neurons, but this effect was not observed during CPP-Test period (Group 4). As shown in Fig. 6i, j (Group 3), compared to the corresponding Veh group, activating the CLGABA neurons that receive projections from mPFC during CPP-Training period effectively reduced the CPP score and ΔCPP score in M-CNO group, and had no effect on CPP score and ΔCPP score in S-CNO group. There was no difference in the total distance traveled among the four groups of mice. However, as shown in Fig. 6k, l (Group 4), when activating these neurons during CPP-Test period compared to the corresponding Veh group, both S-CNO group and M-CNO group exhibited similar CPP score and ΔCPP score. There was no significant difference in the total distance traveled among the four groups of mice.

These results suggest that the top-down mPFC-CL pathway is involved in the encoding association of METH with drug-paired CPP context which was established during METH CPP-Training period, rather than the process of METH reward memory retrieval during METH CPP-Test period.

The bottom-up CL-mPFC pathway contributes to the retrieval of METH reward memory during the process of METH CPP-Test
To explore the role of the CL-mPFC pathway in METH CPP, the bottom-up pathway was regulated during METH CPP-Training period (Group 5 and Group 7) or during METH CPP-Test period (Group 6 and Group 8).

First, inhibiting the CL neurons along the CL-mPFC pathway through chemogenetic regulatory techniques (Fig. 7a, b and Supplementary Fig. 9a, b). As shown in Supplementary Fig. 9c, d, approximately 50% transfection rate in CLGIu neurons and CNO can effectively reduce CL neuronal activity. As shown in Supplementary Fig. 9e, f, compared to the M-Veh group, the M-CNO group during CPP-Test period (Group 6) could effectively reduce the percentage of c-Fos-positive mPFCGlu neurons, but this effect was not observed during CPP-Training period (Group 5). However, the M-CNO group during either CPP-Training period (Group 5) or CPP-Test period (Group 6) had no effect on the percentage of c-Fos-positive mPFCGABA neurons (Supplementary Fig. 9g, h). As shown in Fig. 7c, d (Group 5), when inhibiting the CL neurons projecting to mPFC during CPP-Training period, both S-CNO group and M-CNO group exhibited similar CPP score and ΔCPP score when compared to the corresponding vehicle group. There was no significant difference in the total distance traveled among the four groups of mice. However, as shown in Fig. 7e, f (Group 6), compared to the corresponding Veh group, inhibiting these neurons during CPP-Test period effectively reduced the CPP score and ΔCPP score in M-CNO group, and had no effect on CPP score and ΔCPP score in S-CNO group. There was no difference in the total distance traveled among the four groups of mice. As depicted in Supplementary Fig. 10a–c, when compared to corresponding Pre-Test score, CNO infusion during CPP-Test 1 (on Day 9) significantly reduced CPP score in METH-CNO group mice, and effectively inhibited the activity of Glu neurons within the mPFC (Supplementary Fig. 10d–g) following CNO infusion, indicating that the suppression of CLGlu terminals in the CL-mPFC pathway could block the retrieval of METH reward memory. While, during CPP-Test 2 (on Day 10) that performed after CNO clearance, CPP score was once again elevated relative to CPP-Test 1 levels in METH-CNO group mice, indicating a reactivation of the previously acquired CPP memory (Supplementary Fig. 10c).

Then, we found that the mPFCGABA neurons in the CL-mPFC pathway showed significantly reduced activity during CPP-Test period in METH group mice when compared to the saline group mice, while the mPFCGABA neurons had no significant changes during CPP-Training period (Supplementary Fig. 11a–c). Specifically, we performed chemogenetic manipulation to activate the mPFCGABA neurons along the CL-mPFC pathway (Fig. 7g, h and Supplementary Fig. 12a, b). As shown in Supplementary Fig. 12c, d, approximately 60% transfection rate in mPFCGABA neurons and CNO can effectively enhance mPFC neuronal activity. As shown in Supplementary Fig. 12e, f, compared to the M-Veh group, the M-CNO group during CPP-Test period (Group 8) could effectively reduce the percentage of c-Fos-positive mPFCGlu neurons, but this effect was not observed during CPP-Training period (Group 7). As shown in Fig. 7i, j (Group 7), compared to the corresponding vehicle group, when activating the mPFCGABA neurons that receive projections from CL during CPP-Training period, both S-CNO group and M-CNO group exhibited similar CPP score and ΔCPP score. However, as shown in Fig. 7k, l (Group 8), compared to the corresponding vehicle group, activating these neurons during CPP-Test period effectively reduced the CPP score and ΔCPP score in M-CNO group, and had no effect on CPP score and ΔCPP score in S-CNO group. There was no significant difference in the total distance traveled among the four groups of mice.

These results suggest that the bottom-up CL-mPFC pathway is involved in the retrieval of METH reward memory which was established during METH CPP-Test period, rather than the association process of METH with drug-paired CPP context during METH CPP-Training period.

Methods
Animals
Male C57BL/6 wild-type (WT) mice (6~8 weeks of age, 20~24g) were used (Changzhou Cavens Experimental Animal Co., China). All mice were housed under controlled conditions with constant temperature (24±2°C) and humidity (50±10%) with a 12-h light/dark cycle (light on at 8 a.m.) and allowed free access to food and water in their home cages. All procedures were carried out following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) at Nanjing University of Chinese Medicine, China.

Stereotaxic surgery
Male mice were anesthetized with 2% isoflurane (RWD, China), placed on a stereotaxic apparatus (RWD, China), and their skulls were clamped on both sides of the ears to stabilize the brain. After disinfecting the scalps with iodine tincture followed by 70% medical alcohol, the scalps were incised to expose the skull. A 30% hydrogen peroxide solution was applied to the skull to dissolve the connective tissue, and then microsurgical procedures were initiated. Small holes with a diameter of approximately 1 millimeter were drilled in the skull of the mice to facilitate virus injection or optical fiber implantation. For virus injections, a volume of 200 nL or 50 nL per side was injected into mPFC or CL, respectively. Injections were given over 5 min at a rate of 20 nL/min by an infusion pump (RWD, China) and left in place for 10 min after the procedure. Stereotaxic coordinates were defined as anterior-posterior (AP) from the bregma, mediolateral (ML) from the midline and dorsal-ventral (DV) from the brain surface. The coordinates utilized in this study were as follows, mPFC (AP, +1.94 mm; ML, ±0.4 mm; DV, −2.5 mm), CL (AP, +0.98 mm; ML, ±2.85 mm; DV, −3.85 mm).

Neuron labeling and virus tracing
All viruses utilized in the present study were packaged by BrainVTA (China). For anterograde tracing, the rAAV2/9-hSyn-EGFP-WPRE-hGH pA (PT-1990, 5.24E+12 vg/mL, BrainVTA, China) was unilaterally injected into mPFC or CL of WT mice. For retrograde tracing, the rAAV2/Retro-hSyn-mCherry-WPRE-hGH pA (PT-0100, 5.30E+12 vg/mL, BrainVTA, China) was unilaterally injected into CL or mPFC of WT mice. In order to identify the types of neurons in mPFC that project to CL, the rAAV2/Retro-hSyn-mCherry-WPRE-hGH pA (PT-0100, 5.30E+12 vg/mL, BrainVTA, China) was unilaterally injected into CL. To identify the types of neurons in mPFC that receive projections from the upstream CL, trans-synaptic anterograde virus rAAV2/1-hSyn-CRE-WPRE-hGH pA (PT-0136, 1.09E+13 vg/mL, BrainVTA, China) was unilaterally injected into CL combined with Cre-dependent virus rAAV2/9-hSyn-DIO-EGFP-WPRE-hGH pA (PT-1103, 5.04E+12 vg/mL, BrainVTA, China) in mPFC. Similarly, in order to identify the types of neurons in CL that project to mPFC, the rAAV2/Retro-hSyn-mCherry-WPRE-hGH pA (PT-0100, 5.30E+12 vg/mL, BrainVTA, China) was unilaterally injected into mPFC. To identify the types of neurons in CL that receive projections from the upstream mPFC, trans-synaptic anterograde virus rAAV2/1-hSyn-CRE-WPRE-hGH pA (PT-0136, 1.09E+13 vg/mL, BrainVTA, China) was unilaterally injected into mPFC combined with Cre-dependent virus rAAV2/9-hSyn-DIO-EGFP-WPRE-hGH pA (PT-1103, 5.04E+12 vg/mL, BrainVTA, China) in CL.

Immunofluorescence
Male mice were deeply anesthetized with isoflurane (RWD, China) and sequentially perfused with 0.9% saline and 4% paraformaldehyde (PFA). The brains were removed and post-fixed in 4% PFA at 4°C overnight, then transferred to 30% (w/v) sucrose. The coronal brain sections (30 µm) were continuously sectioned by a cryostat freezing microtome (Leica, Germany) and used for immune-fluorescence. The sections were washed in phosphate-buffered saline (PBS) 3 times (10 min each time) and incubated in 0.3% (v/v) Triton X-100 for 30 min. Then, they were blocked with 5% donkey serum for 90 min at room temperature, and incubated overnight at 4°C with the following primary antibodies: rabbit anti-c-Fos (1:2000, RRID: AB_2247211, Cell Signaling Technology, USA), guinea pig anti-c-Fos (1:3000, RRID: AB_2905595, Synaptic system, Germany), rabbit anti-CaMKII (1:500, RRID: AB_305050, Abcam, UK), rabbit anti-CaMKII (1:500, RRID: AB_2887447, GeneTex, USA), mouse anti-GAD67 (1:500, RRID: AB_2938602, Millipore, USA) followed by the corresponding fluorophore-conjugated secondary antibodies for 90 min at room temperature. CaMKII and GAD67 were used to label Glu and GABA neurons, respectively. The following secondary antibodies were used: Alexa Fluor 555-labeled donkey anti-rabbit secondary antibody (1:500, RRID: AB_162543, Invitrogen, USA), Alexa Fluor 488-labeled donkey anti-rabbit (1:500, RRID: AB_2762833, Invitrogen, USA), Alexa Fluor 555-labeled donkey anti-mouse secondary antibody (1:500, RRID: AB_2762848, Invitrogen, USA), Alexa Fluor 488-labeled donkey anti-mouse secondary antibody (1:500, RRID: AB_141607, Invitrogen, USA), Alexa Fluor 680-labeled donkey anti-rabbit secondary antibody (1:500, RRID: AB_2762836, Invitrogen, USA), Alexa Fluor 680-labeled donkey anti-mouse secondary antibody (1:500, RRID: AB_2762831, Invitrogen, USA), Alexa Fluor 488-labeled donkey anti-guinea pig (1:500, RRID: AB_2736871, Abcam, UK), Alexa Fluor 555-labeled donkey anti-guinea pig (1:500, RRID: AB_3099754, Abcam, UK). Fluorescence signals were visualized using either the Leica DM8I THUNDER Imager 3D Tissue microscope (Leica, Germany) or the Leica TCS SP8 Laser Scanning Confocal microscope (Leica, Germany).

Fluorescence micro-optical sectioning tomography (fMOST)
For fMOST imaging, the rAAV2/9-hSyn-EGFP-WPRE-hGH pA (PT-1990, 5.24E+12 vg/mL, BrainVTA, China) was unilaterally injected into CL and the rAAV2/9-hSyn-mCherry-WPRE-hGH pA (PT-0100, 5.61E+12 vg/mL, BrainVTA, China) was unilaterally injected into mPFC. Three weeks later, male mice were perfused with PBS followed by 4% PFA and brains were dissected and post-fixed in 4% PFA for 24 h at 4°C. After fixation, the brains were rinsed overnight at 4°C in 0.01 M PBS and subsequently dehydrated in a graded ethanol series (25%, 50%, 70% and 100%, 2 h at each concentration) at 4°C. After dehydration, dehydrated brains were put in a brown bottle and infiltrated with 100% LR White mixed with Sudan black B (SBB) Resin (BE-L6-05, OEBio, China) for 9 h. Finally, BioMolding LR White Embedding Kit (BE-L6-05, OEBio, China) was used to polymerize and the special samples were placed in a constant temperature drying chamber at 37.5°C for 24 h. The embedded brains were imaged under an fMOST microscope (BioMapping 5000N, OEBio, China) at a voxel resolution of 0.3×0.3×2 µm. The collected images were processed using Imaris Viewer software (version 9.0, Oxford Instruments, UK).

Fiber photometry
Under physiological conditions, a mixture of the rAAV2/9-EF1α-DIO-hM3D(Gq)-WPRE-hGH pA (PT-2958, 6.53E+12 vg/mL, BrainVTA, China) and the rAAV2/1-hSyn-CRE-WPRE-hGH pA (PT-0136, 5.27E+12 vg/mL, BrainVTA, China) was bilaterally injected into mPFC or CL of WT mice. After 2 weeks of expression, the rAAV2/1-hSyn-CRE-WPRE-hGH pA (PT-0136, 1.09E+13 vg/mL, BrainVTA, China) was bilaterally injected into the mPFC or CL, while the rAAV2/9-VGAT1-DIO-GCaMp6s-WPRE-hGH pA (PT-6173, 5.27E+12 vg/ml, BrainVTA, China) and rAAV2/9-CaMKII-DIO-jRGECO1a-WPRE-hGH pA (PT-2948, 5.32E+12 vg/ml, BrainVTA, China) were bilaterally injected into CL or mPFC. Under pathological conditions, the rAAV2/1-hSyn-CRE-WPRE-hGH pA (PT-0136, 1.09E+13 vg/mL, BrainVTA, China) was bilaterally injected into the mPFC or CL, while the rAAV2/9-VGAT1-DIO-GCaMp6s-WPRE-hGH pA (PT-6173, 5.27E+12 vg/ml, BrainVTA, China) was bilaterally injected into CL or mPFC. An optical fiber (200 µm outer diameter, 0.37 numerical aperture, Thinkerbiotech, China) was placed 100 µm above the viral injection site. After 3 weeks, the calcium-dependent fluorescence signals in mPFC or CL were recorded at their home cages. The calcium-dependent fluorescence signals were obtained by stimulating cells that transfected with the GCaMp6s virus or jRGECO1a virus using a 473 nm LED or 593 nm LED, while calcium-independent signals were obtained by stimulating these cells with a 405 nm LED. The LED lights of 473 nm, 593 nm and 405 nm were alternated at 40 fps and light emission was recorded using sCMOS camera containing the entire fiber bundle (2 min length, NA=0.37, 200 µm core, Thinkerbiotech, China). The analog voltage fluorescent signals were filtered at 30 Hz and digitized at 100 Hz. The signals were recorded and analyzed by ThinkerTech Triple Color Multi Channel fiber photometry Acquisition Software and fiber photometry Analysis Package (Thinkerbiotech, China). For the physiological conditions, F0 is the baseline fluorescence signal which was recorded for 5 min with 0.5-min record and 4.5-min interval (1 session) prior to Clozapine-N-oxide (CNO) treatment. F is the real-time fluorescence signal which was recorded for 60 min with a 0.5-min recording and 4.5-min interval (11 sessions). For the pathological conditions, F0 is the baseline fluorescence signal which was recorded for 0.5 min in home cages. F is the real-time fluorescence signal which was recorded for 15 min in the CPP apparatus. For each session, the photometry signal F was converted to ΔF/F by calculating ΔF/F (%) = (F−F0)/F0×100%. The raw heatmap data from one mouse was merged as a statistical point and normalized using area under the curve (AUC) normalization. The AUC represented the integral under the recording duration relative to the corresponding baseline at each trial.

Acute brain slice preparation
Male mice were deeply anesthetized with isoflurane (RWD, China) and perfused with ice-cold cutting solution (in mM: 92 N-methyl-D-glutamine, 2.5 KCl, 1.2 NaHPO4, 30 NaHCO3, 20 HEPES, 0.5 CaCl2, 10 MgSO4, 25 glucose, 5 sodium ascorbate, 2 thiourea, and 3 sodium pyruvate, pH adjusted to 7.3 with HCl, osmolarity 305, and saturated with 95% O2/5% CO2). Slices containing the mPFC or CL were cut at a 200 µm thickness using a vibratome in 4°C cutting solution. The slices were transferred to 37°C cutting solution and kept for ~9 min, then transferred to oxygenated holding solution (in mM: 86 NaCl, 2.5 KCl, 1.2 NaHPO4, 35 NaHCO3, 20 HEPES, 2 CaCl2, 1 MgSO4, 25 glucose, 5 sodium ascorbate, 2 thiourea, and 33 sodium pyruvate, pH adjusted to 7.3, osmolarity 305, and saturated with 95% O2/5% CO2) to allow for recovery at room temperature for at least 60 min before recordings. During electrophysiological recordings, the brain slices were continuously perfused with oxygenated artificial CSF (ACSF, in mM: 119 NaCl, 2.5 KCl, 1 NaHPO4, 1.3 MgCl2, 2.5 CaCl2, 26.2 NaHCO3, and 11 glucose, osmolarity 290, saturated with 95% O2/5% CO2) and maintained at 31°C by a solution heater (TC-324C, Warner Instruments, USA).

Whole-cell recordings
Whole-cell voltage-clamp microelectrodes (3–5 MΩ) were filled with Cs+-based internal solution (in mM: 130 CsMeSO4, 10 NaCl, 10 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 10 HEPES, pH 7.25–7.30; osmolarity 290). Excitatory postsynaptic currents (EPSCs) were recorded at a holding potential of −70 mV and inhibitory postsynaptic currents (IPSCs) were recorded at a holding potential of +10 mV. A blue light laser was coupled to the stimulator to deliver optostimulation. Optically-evoked EPSCs (oEPSCs) or IPSCs (oIPSCs) were recorded by delivering a 20 Hz pulse train of a 3–5 mW blue laser (473 nm) through an optic fiber which was held upon the target area.

To verify the synaptic connectivity between mPFC and CL, the rAAV2/9-CaMKII-hChR2(H134R)-mCherry-WPRE-hGH pA (PT-0297, 4.78E+12 vg/mL, BrainVTA, China) was bilaterally injected into mPFC and the rAAV2/9-VGAT1-EGFP-WPRE-hGH pA (PT-3176, 5.31E+12 vg/mL, BrainVTA, China) was bilaterally injected into CL. CaMKII-positive neurons in mPFC were identified by mCherry and GABA-positive neurons in CL were identified by EGFP. To verify the synaptic connectivity between CL and mPFC, the rAAV2/9-CaMKII-hChR2(H134R)-mCherry-WPRE-hGH pA (PT-0297, 4.78E+12 vg/mL, BrainVTA, China) was bilaterally injected into CL and the rAAV2/9-VGAT1-EGFP-WPRE-hGH pA (PT-3176, 5.31E+12 vg/mL, BrainVTA, China) was bilaterally injected into mPFC. CaMKII-positive neurons in CL were identified by mCherry and GABA-positive neurons in mPFC were identified by EGFP.

To verify the functional connections of the microcircuits in the mPFC, the rAAV2/1-hSyn-CRE-WPRE-hGH pA (PT-0136, 1.90E+13 vg/mL, BrainVTA, China) was bilaterally injected into CL and the rAAV2/9-VGAT1-DIO-hChR2(H134R)-EYFP-WPRE-hGH pA (PT-4270, 5.13E+12 vg/mL, BrainVTA, China) was bilaterally injected into mPFC. GABA-positive neurons in mPFC were identified by EYFP. To verify the functional connections of the microcircuits in the CL, the rAAV2/1-hSyn-CRE-WPRE-hGH pA (PT-0136, 1.90E+13 vg/mL, BrainVTA, China) was bilaterally injected into mPFC and the rAAV2/9-VGAT1-DIO-hChR2(H134R)-EYFP-WPRE-hGH pA (PT-4270, 5.13E+12 vg/mL, BrainVTA, China) was bilaterally injected into CL. GABA-positive neurons in CL were identified by EYFP.

To examine synaptic specificity, the oEPSCs or oIPSCs were recorded from GABA-positive or CaMKII-positive neurons in voltage-clamp. Tetrodotoxin (TTX, sodium channel blocker, 1 µM) and 4-aminopyridine (4-AP, potassium channel blocker, 100 µM) were used in the recording ACSF solution to verify whether the light evoked currents were monosynaptic. Picrotoxin (PTX, GABA receptor antagonist, 100 µM), 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX, AMPA receptor antagonist, 10 µM) and D-2-Amino-5-phosphonovaleric acid (AP5, NMDA receptor antagonist, 20 µM) were used in the recording ACSF solution to verify neuron-specific connections.

All the light stimulations were applied through QAXK-LASER-473 nm (ThinkerTech, China). All signals were filtered at 4 kHz, amplified at 5× using a MultiClamp 700B amplifier (Molecular Devices, USA) and digitized at 10 kHz with a Digidata 1550B analog-to-digital converter (Molecular Devices, USA). All data were analyzed with Clampfit 10.6 software (Molecular Devices, USA).

Conditioned place preference (CPP)
The conditioned place preference (CPP) test was conducted using a standard two-chambered apparatus equipped with two conditioning compartments, which differed in their patterns (horizontal stripes and vertical stripes) and floors (bar and grid). The experimental paradigm of CPP consisted of three phases: the pre-conditioning test (Baseline/Pre-Test, day 0), conditioning (CPP-Training, day 1–8), and post-conditioning test (CPP-Test, day 9). The mice were habituated to the apparatus 2 days prior to pre-conditioning for 45 min per day. Baseline preference was determined by allowing mice to freely explore both chambers of the CPP apparatus for 15 min. An initial measurement of baseline preference was assessed by recording the time spent in each chamber. Based on initial chamber preference, the non-preferred chamber was designated as the drug-paired chamber, and the preferred chamber was designated as the non-drug-paired chamber. CPP-Training was confined to a non-drug-paired chamber paired with a saline (0.2 mL, i.p.) injection on even days and to a drug-paired chamber paired with METH (1 mg/kg, i.p., China Institute for Food and Drug Control) injection on odd days. After each injection, the male mice were confined to the corresponding chamber (non-drug-paired chamber or drug-paired chamber) for 45 min. During CPP-Test, mice were again placed in the chamber of the CPP apparatus and allowed to explore the two chambers freely without any drug treatment for 15 min.

The CPP score was defined as the time spent in the drug-paired chamber (Tdrug) minus the time spent in the non-drug (used saline as control in the current study)-paired chamber (Tnon-drug) during a given session (Pre-Test or CPP-Test), i.e., CPP score = Tdrug − Tnon-drug. The ΔCPP score was calculated as the CPP score of CPP-Test minus the CPP score of Pre-Test, i.e., ΔCPP score = (Tdrug − Tnon-drug)CPP-Test − (Tdrug − Tnon-drug)Pre-Test. Generally, METH strongly reversed preference and resulted in a positive CPP score, and the CPP score was defined as the extent of the shift in preference after the METH injection.

In vivo chemogenetic manipulation
For chemogenetics regulation of the mPFC-CL pathway, the rAAV2/9-EF1α-DIO-hM4D(Gi)-WPREs-hGH pA (PT-0987, 5E+12 vg/mL, BrainVTA, China) or the rAAV2/1-hSyn-CRE-EGFP-WPRE-hGH pA (PT-1168, 1.07E+13 vg/mL, BrainVTA, China) was bilaterally injected into mPFC, while the rAAV2/Retro-hSyn-Cre-EGFP-WPRE-hGH pA (PT-1186, 5E+12 vg/mL, BrainVTA, China) or rAAV2/9-VGAT1-DIO-hM3D(Gq)-mCherry-WPRE-hGH pA (PT-1725, 5.20E+12 vg/mL, BrainVTA, China) was bilaterally injected into CL. For chemogenetics regulation of the CL-mPFC pathway, the rAAV2/9-EF1α-DIO-hM4D(Gi)-WPRE-hGH pA (PT-0987, 5E+12 vg/mL, BrainVTA, China) or the rAAV2/1-hSyn-CRE-EGFP-WPRE-hGH pA (PT-1168, 1.07E+13 vg/mL, BrainVTA, China) was bilaterally injected into CL, while the rAAV2/Retro-hSyn-Cre-EGFP-WPRE-hGH pA (PT-1186, 5E+12 vg/mL, BrainVTA, China) or rAAV2/9-VGAT1-DIO-hM3D(Gq)-mCherry-WPRE-hGH pA (PT-1725, 5.20E+12 vg/mL, BrainVTA, China) was bilaterally injected into mPFC. CNO (2 mg/kg, i.p., Selleck, USA) was administered 30 min prior to CPP-Training or CPP-Test to inhibit or activate the specific neurons. Saline (0.2 mL) was used as the control vehicle.

For chemogenetics regulation of mPFC terminals in the mPFC-CL pathway and CL terminals in the CL-mPFC pathway, the rAAV2/9-CaMKIIα-hM4D(Gi)-mCherry-WPREs-hGH pA (PT-0050, 5.46E+12 vg/mL, BrainVTA, China) was bilaterally injected into mPFC or CL, and the cannula (mPFC, cat. #62123, O.D. 0.3 mm; CL, cat. #62104, O.D. 0.2 mm, RWD, China) was placed 100 μm above the CL or mPFC. For all cannula mice, CNO (1 mM, 50 nL or 200 nL, Selleckchem, USA) was infused 5 min prior to CPP-Training or CPP-Test via infusion cannulas at a flow rate of 20 nL/min by a syringe pump (RWD, China). Saline (50 nL or 200 nL) was used as the control vehicle.

Data analysis
Statistical analysis was carried out using GraphPad Prism 8.0.2 software. All data were presented as the mean ± SEM. The data were analyzed by unpaired Student’s t test, paired Student’s t test, One-way ANOVA and Two-way ANOVA with Sidak’s multiple comparison test. All statistical significance was set at p < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present a comprehensive study dissecting the bidirectional connectivity between the medial prefrontal cortex (mPFC) and the claustrum (CL), with a focus on their respective roles in the encoding and retrieval of methamphetamine (METH)-associated reward memory. Using a combination of fMOST-based 3D reconstruction, viral tracing, electrophysiology, fiber photometry, and behavioral manipulation in a CPP paradigm, the authors delineate an open-loop reciprocal circuit featuring excitatory and feedforward inhibitory (FFI) pathways. This is an impressive body of work that adds valuable insights to the field of addiction neuroscience and circuit-level reward processing. Following are my specific comments and suggestions:\nMajor Concerns :\n1. The manuscript's textual clarity suffers due to overly dense descriptions. I recommend moving detailed methodological descriptions from the Results to the Methods section, and simplifying circuit descriptions with a better logical flow. Also, removing some abbreviations and references to different cohorts / groups of animals will help. The current format requires the reader to make extensive notes to track pathway-specific findings.\n2. Although fMOST is introduced as a key technique at the very beginning of the results, its analytical contribution is relatively underdeveloped. The 3D reconstruction qualitatively suggests hemispheric projection biases, but lacks quantification. I suggest including: (1) Representative images of AAV spread at the injection site. (2) Quantitative analyses of projection intensities in major downstream targets.(3) Midline thalamic structures are known major mPFC outputs but appear underrepresented in the current image? Could the authors explain a little bit why?\n3. The use of AAV-driven protein expression under VGAT1-promoter to label or manipulate GABAergic neurons in mPFC and CL should be validated. Could the authors provide immunohistochemical co-labeling data (e.g., VGAT1-EGFP with GAD67 or GABA) to assess the specificity and efficiency of targeting in mPFC and CL?\n4. For clarity, please specify which promoters were used in the AAVs driving hM3Dq/hM4Di expression in mPFC and CL during functional manipulations in Figs 6-7 panel A (e.g., CaMKII, hSyn, VGAT).\n5. A novel observation is that mPFC→CL projections are predominantly contralateral, whereas CL→mPFC projections are mainly ipsilateral. The authors may consider functionally testing the necessity of these lateralized connections via unilateral inhibition or disconnection paradigms, or at minimum, expanding the discussion to contextualize the relevance of hemispheric specificity in memory encoding/retrieval.\n6.One of the central findings is the attenuation of feedforward inhibition in both pathways post-CPP. However, the mechanistic underpinnings remain unclear. Future studies or at least a deeper discussion of possible mechanisms would strengthen this interpretation.", "reviewer_2": "This study by Yuhong He et al, presents a comprehensive investigation of the mPFC-CL circuit in METH reward memory. The combination of viral tracing, fiber photometry, chemogenetics, optogenetics, and electrophysiology provides extensive evidence for functionally segregated top-down (mPFC→CL) and bottom-up (CL→mPFC) pathways with distinct roles in encoding vs. retrieval of drug-associated memories. While the finding is interesting, several points require clarification or further consideration:\n1. The results support that the mPFC-CL pathway was engaged during METH CPP memory encoding. The hippocampus has been consistently implicated in contextual memory encoding, especially for the short-term memory. While the PFC is involved in long-term memory regulation and controlling the retrieval of context related memories. The authors need to comment on this issue in more depth.\n2. Chemogenetic/optogenetic manipulations of mPFC or CL neurons projecting to their reciprocal target may activate collateral projections to other reward-related regions (e.g., NAc, hippocampus). How did the authors rule out contributions from these collateral projections to the observed behavioral effects (e.g., CPP scores)? Pharmacological blockade (e.g., local application of glutamate receptor antagonists in CL/mPFC during terminal stimulation) could strengthen the claim that effects are mediated solely by the direct mPFC-CL/CL-mPFC pathways.\n3. It was demonstrated that the bottom-up CL-mPFC pathway contributes to the retrieval of the METH reward memory as shown by the results of inhibiting the CL neurons projecting to mPFC during CPP-Test, not the Training CPP- period by chemogenetic manipulation. If CL-mPFC pathway is responsible for the retrieval, but not the encoding of CPP memory, then CPP memory should be reactivated after the effects of CNO is gone. Further experiments are needed to address this.\n4. The data show FFI is weakened after METH CPP training/expression. Is this weakening a stable trait following conditioning, or does it dynamically change during specific phases (e.g., acquisition, consolidation, retrieval)? Fiber photometry recordings of GABAergic activity specifically during CPP training sessions could address this.\n5. The study implicates GABAergic interneurons in FFI but does not distinguish subtypes (e.g., PV+, SOM+, NPY+). Given known functional heterogeneity (mentioned in Limitations) and evidence that PV/NPY neurons are critical for FFI in PFC/claustrum, could the authors provide data (e.g., post-hoc immunohistochemistry in a subset of experiments) suggesting which GABAergic subtypes mediate the FFI effects observed here? If not feasible, this should be a more prominent limitation.", "reviewer_3": "This is an extensive report exploring the interconnectivity between mPFC and claustrum and their role in mediating methamphehtamine (MA)-associated drug cue memory. Different functional pathways were discovered that mediated the encoding and retrieval of MA CPP memory in mice.\nOverall, this is an interesting and highly novel study. The experimental design was solid and the writing was clear. The results were clear and the conclusion was supported by the data. I have no major conners.\nModerate to minor concerns:\n1. The CPP score and deltaCPP score were not clearly described. It seems that the deltaCPP score, which was calculated by subtracting CPP Pre-test score from CPP Test score. However, it is not clear how this was calculated: the METH-paired side only? Because CPP score was time spent in drug-paired chamber minus time spent in saline-paired chamber, does deltaCPP score mean=CPP test score - pre-test score = (time spent in drug-paired side - time spent in saline-paired side during CPP test) - (time spent in drug-paired side - time spent in saline-paired side during CPP pre-test)? Because the total time during pre-test and test was 900 seconds (15 min), the above calculation would get: deltaCPP score = 2 (time spent in drug-paired chamber during test - time spent in drug-paired chamber during pretest). This is not what CPP scores are typically calculated because this essentially doubled (2X) the actual drug-induced CPP preference. This needs to be clearly articulated.\n2. There are some minor grammatical errors that need to be fixed."}, "author_response": "Response: Thank you for your suggestion. We have revised the issue throughout the manuscript to improve textual clarity by: (1) relocating detailed methodological descriptions from the Results to the Methods; (2) simplifying circuit descriptions to enhance logical flow and readability; (3) reducing abbreviations and minimizing references to multiple animal cohorts/groups.\nResponse: Thank you for these constructive suggestions. We have revised the manuscript according to your suggestions.\n(1) As shown in Fig 1 A1 and Fig 1 B1, we have added the representative images of the injection sites to verify the accuracy and reliability of viral delivery (Extended Data Figure 1 A).\n(2) To strengthen the analytical contribution of the fMOST data, we have now included quantitative analyses of projection signal intensities across downstream brain regions of either mPFC or CL (Fig 1 A2-A3 and Fig 1 B2-B3).\n(3) The weaker mPFC projection signals to midline thalamic nuclei in our 3D reconstructions may be attributable to laminar specificity: mPFC neurons projecting to midline thalamic nuclei arise mainly from layer 6 1, 2, whereas those projecting to the CL are primarily located in layer 5 3. In the present study, the tracing virus was injected into the mPFC (coordinates from bregma: AP, + 1.94 mm; ML, +/- 0.4 mm; DV, - 2.5 mm). As shown in Fig 2A, the virus predominantly transfected layer 5 neurons, as indicated by the quantification of fluorescence intensity at the mPFC injection site, which likely explains the relatively weaker axonal signals observed in the mediodorsal thalamic nucleus (MDT, located in the midline thalamic nuclei) in our 3D reconstructions (Figure 1 A2).\nResponse: Thank you for raising this important point. In our study, we performed immunofluorescence co-labeling of mCherry-expressing neurons (AAV2/9-DIO-VGAT-hM3D(Gq)-mCherry) with the GABAergic marker GAD67, along with c-Fos to assess activity-dependent expression. These results are presented in the original supplementary figures (Extended Data Figure 8 A3-A4 for the CL and Extended Data Figure 12 A3-A4 for the mPFC), and show a high degree of overlap between VGAT-labeled neurons and GAD67-positive cells, demonstrating specific targeting of GABAergic populations. Moreover, the strong expression of c-Fos in mCherry-positive neurons further supports effective viral transfection. Together, these data confirm the specificity and reliability of our approach for selectively labeling and manipulating GABAergic neurons in both the mPFC and CL.\nResponse: Thank you for your comments. In the experiments shown in Figures 6 A and 7 A, the AAVs driving hM4Di expression in the mPFC and CL employed the EF1α promoter. As you suggested, we have added this information directly to Figures 6 A and 7 A for clarity. Full details of the viral constructs and catalog numbers are also provided in the Methods section.\nResponse: Thank you for your suggestion.\n(1) Since most of our experiments were based on in vitro recordings and staining in brain slices, it was difficult to distinguish hemispheric specificity in functional manipulations within the current timeframe. However, your comment has prompted us to consider this important dimension for future studies, where hemisphere-specific inhibition or disconnection approaches could be applied.\n(2) As you suggested, we have expanded the Discussion section to place our findings in the context of hemispheric specialization. The added text is as follows: “In this study, we observed hemispheric asymmetry in the connectivity pattern between mPFC and CL, as indicated that mPFC mainly send projections to the contralateral CL, whereas CL projections predominantly send projections to the ipsilateral mPFC. Although the cerebral hemispheres are structurally similar, they exhibit functional lateralization in cognitive processes, including memory. Some studies have demonstrated that different types of memory information are predominantly encoded by distinct hemispheres. For instance, the left PFC is more strongly implicated in episodic memory encoding, while the right PFC is preferentially engaged during episodic memory retrieval4. Such specificity of the left and right hemispheres in memory encoding/retrieval is not absolute but rather involves mutual collaboration and dynamic changes. Studies have reported that the left and right hemispheres are interconnected by abundant nerve fiber pathways, such as the corpus callosum, enabling information communication and integration during memory-related processes5. These perspectives raise the intriguing possibility that the mPFC-CL interconnectivity might contribute to METH-related memory encoding and retrieval through hemisphere-specific manipulations.”\nResponse: Thanks a lot. Following your suggestion, we have expanded the Discussion section to address potential mechanisms underlying the attenuation of feedforward inhibition (FFI) over METH CPP.\n(1) Previous work has shown that cortical inputs to the CL evoke robust FFI within the CL6, suggesting that the cortex not only excites but also exerts selective inhibitory control over claustral circuits through the recruitment of local interneurons. In this study, we identified both a direct mPFCGlu-CLGlu excitatory pathway and an indirect mPFCGlu-CLGABA-CLGlu inhibitory pathway, which together may gate cortico-claustral transmission by balancing excitation and inhibition. We also found that activation of mPFC could suppress CL under normal condition, while both mPFC and CL were activated after METH CPP in male mice. These results suggest that METH may reduce inhibitory tone of CLGABA neurons on CLGlu neurons, resulting in both mPFC and CL run toward excessive excitation.\n(2) As to the bottom-up CL-mPFC pathway, electrophysiological studies demonstrate that CL inputs predominantly induce strong and widespread FFI in the PFC under physiological conditions, largely via parvalbumin (PV+) and neuropeptide Y (NPY+) interneurons7. This inhibitory control ensures precise regulation of prefrontal excitability and prevents runaway excitation. We speculate that METH disrupts this inhibitory brake by impairing the indirect CLGlu-mPFCGABA-mPFCGlu inhibitory pathway, ultimately weakening the inhibitory regulation of mPFC outputs.\n(3) METH exposure is known to remodel the excitatory-inhibitory (E/I) balance by impairing the interneurons’ excitability8. Here, we found that weakened FFI, caused by impaired local interneurons within the indirect pathways of mPFC-CL circuits, represents a key mechanism underlying METH-induced CPP. The reduction in inhibitory gating of CLGlu neurons by CLGABA interneurons likely impairs the CL’s ability to filter cortical inputs, leading to excessive propagation of excitatory signals. In turn, weakened CL-driven FFI on mPFCGABA-mPFCGlu would diminish inhibitory control over prefrontal outputs, amplifying excitatory drive during the retrieval of drug-associated memories. This bidirectional breakdown in mPFC-CL circuits could reinforce drug cues and impair cognitive flexibility, which contribute to the persistence of maladaptive drug-context associations. Besides, we also found that the weakened FFI in the top-down mPFC-CL pathway and in the bottom-up CL-mPFC pathway may contribute to different stages of METH-induced reward memory. Specifically, disinhibition of FFI of mPFCGABA neurons on mPFCGlu neurons could rescue the retrieval of METH reward memory instead of encoding phase of METH-context association. Conversely, strengthen of FFI of CLGABA neurons on CLGlu neurons suppress the initial formation of METH reward memory rather than the retrieval phase. This phenomenon may be associated with the reciprocal architecture between mPFC and CL, which recruiting different patterns of information communication and integration in the brain. Overall, the mPFC-CL circuits contribute to integrate drug-associated contextual memories in a pathway-specific manner, potentially facilitating their persistence and retrieval during METH-related behaviors.\nIn the future, several lines of investigation may clarify the role of FFI attenuation in METH CPP. First, cell-type-specific approaches are needed to determine whether PV+ neurons, somatostatin (SOM+) neurons, vasoactive intestinal peptide (VIP+) neurons and NPY+ neurons are preferentially affected in the CL and mPFC. Second, molecular analyses of GABA release and GABA receptor subunits at inhibitory synapses may help identify pharmacological entry points.\nResponse: Thank you for your comments. Following your suggestion, we have expanded the Discussion section to address the complementary roles of the hippocampus and prefrontal cortex (PFC) in memory processes. “Notably, the process of encoding and retrieval of reward memories require the coordinated activity across multiple brain regions. The hippocampus is essential for contextual memory encoding during the early phases of short-term memory formation1, 2, rapidly stabilizing environmental cues linked to reward or aversion3, 4. By contrast, the mPFC plays a more prominent role in the regulation and retrieval of long-term contextual memories, as well as in the executive control of memory-guided behaviors5. Preston et al. have reported that the progress of initial memory to stabilized ones become less dependent on the hippocampus, but rely much on neocortical networks6. In the current study, we found that the mPFC-CL pathway is engaged in the encoding process of METH-related reward memory, indicating that mPFC-CL pathway might coordinate with hippocampus to progressively assist subsequent cortical consolidation. As mentioned above, the mPFC-CL pathway might contribute to the selection and suppression of some cortical ensembles, thereby refining which of contextual associations become stabilized into long-term memory. In the future study, the role of network among hippocampus, mPFC and CL in the drug-related reward memory should be further explored.”\nResponse: Thank you for your comments. As you mentioned, drug-related memory is much complicated progress that recruit many regions or nuclei in the brain, forming a complex underlying network. In the current study, we focus on the role of mPFC-CL circuits in the encoding and retrieval of METH reward memory. We believe that mPFC-CL circuits are not the only mechanism involved in METH reward memory and must work in coordination with other brain regions. In the current study, we acknowledge that chemogenetic or optogenetic manipulations of mPFC or CL neurons could potentially affect collateral projections to other reward-related regions, such as the NAc or hippocampus. To minimize this issue, we employed a dual viral-vector strategy that selectively target projection-specific populations (mPFC-CL or CL-mPFC). This approach has been widely validated in many other studies involving the mPFC- and CL-related pathway regulation7, 8. While such targeting substantially reduces off-target effects, we tried our best but cannot fully exclude the possibility of collateral contributions.\n(1) To further address this concern, we performed additional experiments (Extended Data Figures 6 A and 10 A) using a projecting terminal chemogenetic inhibition strategy. Specifically, we injected an anterograde AAV encoding CaMKII-hM4D(Gi) into the mPFC or CL and implanted local cannula into the reciprocal target (CL or mPFC). This design enabled local infusion of CNO directly into the CL or mPFC, thereby selectively inhibiting presynaptic terminals from mPFC or CL within CL or mPFC, minimizing effects on collateral projections.\n(2) As shown in Fig 3A, chemogenetic inhibition of mPFCGlu terminals in the CL during CPP-Training period blocked the acquisition of METH CPP (Extended Data Figure 6 A1-A4) and effectively suppressed the activity of glutamatergic neurons in the CL (Extended Data Figure 6 A5-A8) following CNO infusion.\n(3) As shown in Fig 4A, chemogenetic inhibition of CLGlu terminals in the mPFC during CPP-Test 1 period suppressed the acquisition of METH CPP (Extended Data Figure 10 A1-A3), and effectively inhibited the activity of glutamatergic neurons within the mPFC (Extended Data Figure 10 A4-A7) following CNO infusion.\n(4) Together, these additional experiments strengthen our conclusion that the mPFC-CL pathway and CL-mPFC pathway play important role in encoding and retrieval of METH reward memory, respectively.\n(5) We have incorporated this new data (Extended Data Figures 6 A and 10 A) in the revised manuscript.\nResponse: Thank you for your comment. To further clarify whether the CL-mPFC pathway contributes specifically to reward memory retrieval, we conducted follow-up CPP tests (CPP-Test 2) after CNO clearance (Fig 4 A).\n(1) As depicted in Fig 4 A3, when compared to corresponding Pre-Test score, CNO infusion during CPP-Test 1 (on Day 9) significantly reduced CPP score in METH-CNO group mice, indicating that the suppression of CLGlu terminals in the CL-mPFC pathway could block the retrieval of METH reward memory. While, during CPP-Test 2 (on Day 10) that performed after CNO clearance, CPP score was once again elevated relative to CPP-Test 1 levels in METH-CNO group mice, indicating a reactivation of the previously acquired CPP memory. These findings suggest that inhibition of CLGlu terminals in the mPFC during CPP-Test period specifically disrupted memory retrieval rather than memory encoding.\n(2) We have incorporated this new data as Extended Data Figure 10 A in the revised manuscript.\nResponse: Thank you for raising the important questions. To address this point, we performed fiber photometry recordings of GABAergic neuronal activity throughout the entire METH CPP protocol (Extended Data Figures 7 A and 11 A), which are described in the Results section and Discussion section.\n(1) As shown in Fig 5 A and Fig 6 A, both CLGABA neurons along mPFC-CL pathway and mPFCGABA neurons along CL-mPFC pathway exhibited significantly reduced activity during either CPP-Training period or CPP-Test period in METH group mice, respectively. These reductions occurred in the same time windows in which chemogenetic/optogenetic manipulations produced behavioral effects in our previous experiments. However, as shown in Fig 5 A3, the CLGABA neurons activity had no significant changes during CPP-Test period. In parallel, the mPFCGABA neurons activity had no significant changes during CPP-Training period.\n(2) Specifically, disinhibition of mPFCGABA neurons is not required for initial learning but is progressively recruited as the METH-context association strengthens and becomes critical for the retrieval of reward memory. By contrast, disinhibition of CLGABA neurons is not essential for reward memory retrieval but appears to be engaged in the initial formation of reward memory. This stage-specific modulation may reflect the reciprocal architecture of the mPFC-CL circuits, which facilities specific information communication and integration in pathway-specific manner.\nResponse: Thanks for your suggestion.\n(1) In the present study, we focused on GABA-positive neurons without further subdivision into specific subtypes. As to your suggestion, we tied to stain PV+ neurons, SOM+ neurons and NPY+ neurons with the VGAT-transfected positive neurons in CL along the mPFC-CL pathway or mPFC along CL-mPFC pathway following the fiber photometry recordings throughout the entire METH CPP protocol (Fig 5 A1 and Fig 6A1). This data included the available staining results in the response letter, instead of putting these data into resubmission, partially due to unsatisfactory immunostaining results for certain markers.\nAs shown in Fig 7A1-A3, it was about 3.3 of PV+ neurons, 5.6 of SOM+ neurons and 14.2 of NPY+ neurons co-labeled with VGAT-positive neurons in METH group mice during METH CPP-Training period. Similarly, as shown in Fig 7B1-B3, it was about 1.6 of PV+ neurons, 1.2 of SOM+ neurons and 8.3 of NPY+ neurons co-labeled with VGAT-positive neurons in METH group mice during METH CPP-Test period.\n(2) We have emphasized this limitation in the Discussion section: “① Second, our study did not distinguish GABAergic interneuron subtypes within the mPFC-CL circuits, despite emerging evidence suggesting substantial functional heterogeneity among them9, 10, 11. In mPFC, GABAergic neurons include parvalbumin (PV) neurons, somatostatin (SOM) neurons, vasoactive intestinal peptide (VIP) neurons and neuropeptide Y (NPY) neurons. Specifically, PV and NPY are thought to be central to FFI12, 13, 14. In CL, GABAergic neurons are predominantly divided into PV and SOM subtypes15, with PV neurons likely playing a crucial role in FFI14, 16. ② In future work, several lines of investigation may clarify the role of FFI attenuation in METH CPP. First, cell-type-specific approaches are needed to determine whether PV+ neurons, SOM+ neurons, VIP+ neurons and NPY+ neurons are preferentially affected in the CL and mPFC.”\nResponse: Thank you for your comments.\n(1) CPP score was defined as the time spent (T) in the drug-paired chamber minus the time spent in the non-drug (used saline as control in the current study)-paired chamber during a given session (Pre-Test or CPP-Test), i.e., CPP score = T − T .\ndrug non-drug\n(2) ΔCPP score was calculated as the CPP score of CPP-Test minus the CPP score of Pre-Test, i.e., ΔCPP score = (T − T ) − (T − T\ndrug non-drug CPP-Test drug non-\ndrug Pre-Test.\nThe ΔCPP score represents the change in preference for the drug-paired chamber induced by conditioning, rather than a doubling of time spent in the drug-paired chamber.\n(3) We have revised the Methods section to present the formula explicitly and ensure that the calculation procedure is clear.\nResponse: Thank you for your suggestion. We have tried our best to revise the resubmission thoroughly."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a great job addressing my previous concerns. I can now recommend for publication. Congrats on this exciting work!", "reviewer_2": "The authors have undertaken a substantial revision of their manuscript, providing extensive new data and clarifications that significantly strengthen the study. The additional experiments—particularly the terminal-specific chemogenetic manipulations, longitudinal fiber photometry of GABAergic activity, and follow-up CPP tests—effectively address several key concerns raised during the initial review. The manuscript now presents a more compelling and nuanced account of the mPFC–CL circuit's role in METH reward memory.\nThe authors provided preliminary co-labeling data for VGAT+ neurons with PV, SOM, and NPY (Fig. 7). While the low co-expression percentages (e.g., ~3-14%) are interesting, they are difficult to interpret without knowing the baseline proportions of these subtypes in the total VGAT+ population. Could the authors provide reference data for the expected distribution of PV+/SOM+/NPY+ neurons among CL and mPFC GABAergic cells?\nThe authors have included the \"male mice\" in the manuscript title, which indicating that the findings are derived from a single-sex study. While the authors mention in the limitations that \"hormonal fluctuations\" in females could be a confounding factor, the implications of the single-sex design for the generalizability of the conclusions should be explicitly stated in the discussion.", "reviewer_3": "The revision addressed my previous concerns. I have no more concerns."}, "author_response": "Response: Thanks for your review.\nResponse: Thank you for your comments.\n(1) According to previous studies, approximately 20% and 30% of GABAergic neurons are immunoreactive for PV+ and SOM+ in the CL, respectively 1. Although direct quantitative data on NPY+ neurons in the CL are limited, existing evidence suggests that their proportion is substantially lower than that of PV+ and SOM+ neurons 2. In the cerebral cortex, PV+, SOM+, and NPY+ interneurons each constitute roughly 40%, 30% and 10% of the total GABAergic population 3, 4, respectively.\n(2) In our present study, approximately 3.3% of PV+, 5.6% of SOM+ and 14.2% of NPY+ neurons co-labeled with VGAT-positive neurons which receiving projection from the mPFC in the CL, whereas 1.6% of PV+, 1.2% of SOM+ and 8.3% of NPY+ neurons co-labeled with VGAT-positive neurons which receiving projection from the CL in the mPFC. The relatively low co-expression levels may be attributed to methodological factors, such as antibody sensitivity and labeling efficiency. However, these results do not affect our conclusion and innovativeness of our findings.\n(3) In future work, we plan to further investigate the specific roles of GABAergic subtypes within the mPFC-CL circuits in METH-related reward memory using Cre-driver mouse lines for cell-type–specific manipulation.\nResponse: Thank you for your suggestion. We have emphasized this limitation in the Discussion section: First, there is lack of female mouse model, partly due to the known effects of the estrous cycle and hormonal fluctuations on neuronal excitability and behavioral outcomes 5, 6, 7. Previous human and animal studies have shown that females are more susceptible to developing and maintaining addiction as well as relapse induction compared to males, and these sex differences are largely attributed to the modulatory effects of estrogen on the dopaminergic system 8, 9. In the current study, we only used male mice to explore the role of mPFC-CL circuits in METH-related reward memory. Whether similar neural and behavioral mechanisms occur in female mice remains to be determined, and this issue will be an important focus of future research.\nResponse: Thanks for your review."}]

Decision letter