Introduction

Alcohol use disorder (AUD) is a chronic, relapsing brain condition affecting over 14 million adults in the U.S., characterized by compulsive alcohol consumption, impaired control over drinking, and negative emotional states during withdrawal. Stress is a key contributor to both the development and recurrence of AUD, with stressful experiences and elevated stress hormone levels frequently precipitating relapse episodes in abstinent individuals [1–3]. An expanding body of clinical and preclinical evidence highlights the importance of stress-responsive neurocircuits in driving addiction-related behaviors, implicating neuropeptides like corticotropin-releasing factor (CRF) in relapse vulnerability [4–8]. CRF is a central regulator of the stress response, coordinating hormonal and behavioral adaptations to stress through widespread action in both hypothalamic and extrahypothalamic brain regions. In addition to initiating hypothalamic-pituitary-adrenal (HPA) axis activity, CRF modulates affective and motivational processes through its action within the amygdala, bed nucleus of the stria terminalis (BNST), and other limbic structures.

Substantial evidence indicates that CRF signaling promotes drug-seeking behavior during stress and withdrawal across multiple substances of abuse, supporting its role as a critical mediator of relapse [5, 6, 9]. Despite extensive research on CRF in limbic areas, its role in the dorsal striatum, an area crucial for habit formation and behavioral flexibility, remains less well understood [10–16]. Within this region, cholinergic interneurons (CINs) are key regulators of striatal output and acetylcholine (ACh)-mediated dopamine modulation, integrating diverse inputs and contributing to reward-based learning [17–19]. CINs are sensitive to neuromodulatory influences, yet it is unclear whether they are directly targeted by CRF and how this interaction might be altered by alcohol exposure [20–22].

In this study, we investigated a novel CRF-CIN circuit linking the CeA and BNST to the dorsal striatum. Using monosynaptic and retrograde circuit tracing, we identified direct projections from CRF-expressing neurons to dorsal striatal CINs. CRF enhanced CIN excitability and promoted ACh release via CRFR1 activation, but this excitatory effect was disrupted by acute alcohol exposure, indicating that alcohol interferes with CRF-dependent cholinergic modulation. These findings identify a CRF-CIN circuit that is vulnerable to alcohol-induced dysregulation, providing mechanistic insight into how stress peptides and alcohol interact to impair striatal function.

Materials and Methods

Animals

Male and female 3-4-month-old mice or rats were used in all studies. Rats were mainly used for histological validation of CRFR1 expression with the CRFR1-Cre-tdTomato line, whereas mice were used for histology, electrophysiology, optogenetics, and GRAB-ACh sensor experiments because of the availability of transgenic Cre-driver and reporter lines. ChAT-eGFP (stock 007902), ChAT-Cre (stock 031661), Drd1a-tdTomato (D1-tdT, stock 016204), Ai32 (stock 012569), CRH-ires-CRE (stock 012704) and C57BL/6J (stock 000664) mice were purchased from The Jackson Laboratory [23–27]. CRFR1-GFP mice were gifted by Dr. Marisa Roberto’s lab. All mice were backcrossed onto a C57BL/6 background. ChAT-Cre mice were crossed with D1-tdTomato mice to obtain ChAT-Cre;D1-tdTomato mice. CRF-Cre mice were crossed with ChAT-eGFP mice to generate CRF-Cre;ChAT-eGFP mice. CRF-Cre;Ai32 mice were generated in-house and crossed with ChAT-eGFP to generate triple transgenic CRF-Cre;Ai32;ChAT-eGFP mice. We used CRFR1-Cre-2A-tdTomato rats that have been validated for expression of Cre and tdTomato in CRFR1-expressing neurons [28]. We also used CRF-Cre rats that have been shown to express Cre recombinase in CRF-producing neurons in the CeA and BNST [29]. We crossed CRF-Cre rats with a Cre-dependent tdTomato reporter line to visualize CRF⁺ neurons. Genotypes were confirmed through PCR analysis of tail DNA to detect Cre or fluorescent protein genes in mice and rats (Cre for CRF-Cre and ChAT-Cre, tdTomato for D1-tdTomato, and GFP for Ai32) [30–34]. Animals were housed in a temperature- and humidity-controlled vivarium with a 12-h light/dark cycle. Food and water were available ad libitum. The Texas A&M University Institutional Animal Care and Use Committee approved all animal care and experimental procedures.

Reagents

AAV8-Ef1a-FLEX-TVA-mCherry (lot # AV5008b), AAV8-FLEX-RG (lot # AV5005f), and AAV-FLEX-Chrimson-tdTomato (lot # AV5844) were purchased from the UNC Vector Core [35, 36]. AAVrg-pCAG-FLEX-eGFP-WPRE (catalog # 51502-AAVrg) was purchased from Addgene [37], while EnvA-SADΔG-GFP was purchased from the Salk Institute [38]. Choline acetyltransferase (AB144P) antibody was purchased from Sigma [39]. ACh sensor (AAV-GRAB_ACh4m) was obtained from BrainVTA (PT-7021) [40–44], which is an updated version of ACh3.0 [45]. CRF peptide and NBI 35695 (CRFR1 antagonist) were obtained from Tocris. 6,7-dinitroquinoxaline-2,3-dione (DNQX), tetrodotoxin (TTX), 4-aminopyridine (4-AP), and bicuculline were also purchased from Tocris.

Stereotaxic virus infusion

The stereotaxic virus infusion procedure was conducted as described previously [30, 46, 47]. When required for the experimental design, AAV-DIO-TVA-mCherry and AAV-DIO-RG were bilaterally infused into the dorsomedial striatum (AP: 0.38 mm, ML: ±1.55 mm, DV: -2.90 mm from the Bregma) [48] of ChAT-Cre;D1-tdTomato mice (Fig. 1). Rabies-GFP was infused at the same injection site three weeks later at a 10-degree angle to avoid contamination of the infusion tract. Rabies-GFP virus was allowed to incubate for one week. AAVrg-pCAG-FLEX-eGFP-WPRE was infused into the dorsal striatum (AP: 0.00, mm, ML: ±2.80 mm, DV: -4.85 mm from the Bregma) [49, 50] of CRF-Cre;tdTomato rats (Fig. 3A-3E). Additionally, AAV-DIO-Chrimson-tdTomato was infused into the CeA (AP: -1.60 mm; ML: ±4.20 mm; DV: -8.10 mm) and BNST (AP: -0.10 mm; ML: ±1.40 mm; DV: -6.70 mm) of CRF-Cre;ChAT-GFP mice as described in published literature [51] (Fig. 3F-3H). The animals were placed on a stereotaxic surgical frame after being sedated with 3-4% isoflurane at a rate of 1.0 L/min, as described previously [46, 47, 52]. These coordinates were obtained from previous publications and verified using the rat [53] or mouse [54] brain atlases. A volume of 0.5 µL/site (mice) or 1 µL/site (rats) of virus was infused at a rate of 0.08 µL/min. At the end of the infusion, the injectors remained at the injection site for 10 to 15 min before removal to allow for virus diffusion. The scalp incision was then sutured and animals were returned to their home cage for recovery.

Figure 1.

Histology and cell counting

Animals were anesthetized and perfused intracardially with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). The brains were extracted and submerged in 4% PFA/PBS solution for one day at 4°Cbefore being transferred to a 30% sucrose solution in PBS. Once the brains completely sank in the sucrose solution, they were cut into 50-µm thick coronal sections using a cryostat. The slices were stored in a PBS bath at 4°C before mounting on slides for imaging using a confocal laser-scanning microscope (Fluoview, Olympus). All images were processed using Imaris 8.3.1 (Bitplane, Zurich, Switzerland) as previously reported [30, 32, 34]. Immunostaining for choline acetyltransferase (ChAT) was performed using an anti-ChAT primary antibody (AB144P), followed by a fluorophore-conjugated secondary antibody (A21447) emitting at 647 nmto label CINs with far-red fluorescence. Cell counting was performed in 8 CRF-Cre;tdTomato rats. For each brain region, 5-10 brain sections were imaged from each animal. Imaris was used to quantify green and red neurons as well as evaluate colocalization. Brain regions were identified using the Mouse Brain Atlas [54]. Images were primarily obtained from the posterior dorsomedial striatum, corresponding to coronal slices posterior to the crossing of the anterior commissure and anterior to the tail of the striatum (starting around 0.62 mm and ending at −1.3 mm relative to the Bregma).

Slice electrophysiology

Slice preparation

Slices were prepared and electrophysiological recordings were conducted as described previously [30, 32, 46, 47, 55]. Briefly, coronal sections (250 μm) containing the posterior dorsomedial striatum were cut in an ice-cold cutting solution containing (in mM): 40 NaCl, 148.5 sucrose, 4 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 0.5 CaCl₂, 7 MgCl₂, 10 glucose, 1 sodium ascorbate, 3 sodium pyruvate, and 3 myo-inositol. The solution was saturated with 95% O₂ and 5% CO₂. Slices were then incubated in a 1:1 mixture of the cutting and external solutions at 32°C for 45 min. The external solution was composed of the following (in mM): 125 NaCl, 4.5 KCl, 2.5 CaCl₂, 1.3 MgCl₂, 1.25 NaH₂PO₄, 25 NaHCO₃, 15 glucose, and 15 sucrose. The external solution was saturated with 95% O₂ and 5% CO₂. Slices were then maintained in the external solution at room temperature until use. Recordings were primarily obtained from the posterior dorsomedial striatum, corresponding to coronal slices posterior to the crossing of the anterior commissure and anterior to the tail of the striatum (starting around 0.62 mm and ending at −1.3 mm relative to the Bregma). More anterior slices were occasionally included to increase the sample size.

Cell-attached recordings

Individual slices were transferred to a recording chamber and continuously perfused with the external solution at 2-3 mL/min at 32°C. Fluorescent axonal fibers and neurons were visualized using an epifluorescence microscope (Olympus). The presence of tdTomato and GFP allowed cell type verification and expression of Ai32 allowed visualization of CRF-producing fibers. CINs in slices were identified by their labeled color, large size, and spontaneous firing. Spontaneous cell-attached CIN firing activity was recorded for 5 min to calculate the average firing frequency. Light at 470-nm or 590-nm light was delivered from the objective lens for 2 ms at frequencies and durations specified in the figure legends to stimulate channelrhodopsin-expressing axonal fibers in the dorsal striatum. We used a potassium-based intracellular solution containing (in mM): 123 potassium gluconate, 10 HEPES, 0.2 EGTA, 8 NaCl, 2 MgATP, and 0.3 NaGTP, with an osmolarity of ∼280 mOsm/L and the pH adjusted to 7.3 using KOH. For protocols involving alcohol application, we pretreated dorsal striatal slices with alcohol (50 mM) for 1 h outside of the recording chamber. After pretreatment, slices were transferred to the recording chamber, where alcohol was washed out for at least 15 min before recording. The recordings included a 10-minute baseline period (without alcohol), followed by 10 minutes of CRF bath application and 10 minutes of alcohol exposure. Control slices did not receive alcohol pretreatment but were exposed to acute alcohol following CRF application. In both conditions, the final 10-min alcohol application allowed us to assess whether alcohol pretreatment altered the CIN firing response to subsequent CRF and alcohol exposure. In protocols that required inhibition of glutamatergic and GABA_A receptors, DNQX and bicuculline were used, respectively. Electrophysiology data were acquired using Clampex-10 (Molecular Devices) and analyzed using Clampfit-10 (Molecular Devices) and Mini Analysis (Mini60, Synaptosoft Inc.).

Ex vivo live-tissue confocal imaging of ACh release

Brain slices were kept in a recording chamber perfused with oxygenated external solution (95% O₂ and 5% CO₂). An Olympus FluoView FV3000 microscope was used with a 10x NA 0.3 and a 40x NA 0.8 water immersion objective, along with a 488 nm and a 561 nm laser. The sample rate of imaging was 2 to 3 frames per second. Parameters were maintained consistently across all imaging sessions, including laser intensity, HV, gain, offset, and aperture diameter.

Statistical analysis

Before conducting all experiments shown in this study, we performed a power analysis with SigmaPlot software (12.5, Systat) using the mean and standard deviation from previous studies in our lab [34, 48–50] to determine the required sample sizes to detect a significant difference. We tested all data for normality before significance testing. If the normality test failed, we used nonparametric tests, such as the Mann-Whitney U Test. All data are expressed as mean ± SEM. Data were analyzed by two-tailed t-test (unpaired or paired), one- or two-way ANOVA with repeated measures, followed by the Tukey or Sidak post hoc test. Significance was determined if p < 0.05. Statistical analysis was conducted by the SigmaPlot program. Graphs were constructed using the OriginPro (2024b, OriginLab) program.

Results

Dorsal Striatal CINs Receive Monosynaptic Inputs from CeA and BNST Neurons

To investigate the connection between dorsal striatal CINs and stress-related brain regions, we examined whether CINs receive monosynaptic inputs from the CeA and the BNST [56, 57]. We used ChAT-Cre;D1-tdTomato mice to perform rabies-mediated monosynaptic retrograde tracing. In this mouse model, CINs express Cre recombinase, and dopamine D1-receptor (D1R)-expressing medium spiny neurons (D1-MSNs) are labeled with tdTomato. Including the D1-tdTomato marker allowed us to delineate the CeA and BNST, which, unlike surrounding striatal areas, do not express D1Rs [52]. We performed rabies-mediated retrograde monosynaptic circuit tracing (Fig. 1A), allowing specific targeting of CINs (Fig. 1B). In addition to labeling neurons in brain regions known to project to dorsal striatal CINs—including the striatum itself, cortex (e.g., cingulate cortex, motor cortex, somatosensory cortex), thalamus (e.g., parafascicular thalamic nucleus, centrolateral thalamic nucleus), globus pallidus, and midbrain—we were surprised to see labeled neurons in both the CeA (Fig. 1C) and BNST (Fig. 1D), which are well-characterized as key stress-responsive nuclei. Interestingly, the BNST had a higher density of DMS-projecting neurons than the CeA (Fig 1E; Mann-Whitney U = 4.000, ***p < 0.001). These results demonstrate that dorsal striatal CINs receive direct, monosynaptic inputs from neurons in the CeA and BNST. Together, these findings highlight the anatomical connection of dorsal striatal CINs with key stress-responsive brain regions.

The Dorsal Striatum Lacks CRF⁺ Neurons Unlike the CeA and BNST

We first investigated whether the dorsal striatum contains CRF-producing cells or whether CRF signaling in this region depends on CeA and BNST inputs. While previous studies have identified CRF-producing neurons in the CeA and BNST [51], their presence in the dorsal striatum was uncertain. Coronal brain sections from CRF-Cre;tdTomato rats revealed dense populations of CRF-tdTomato⁺ neurons in the CeA (Fig. 2A) and BNST (Fig. 2B). Although CRF⁺ axonal fibers were observed in the dorsal striatum, CRF⁺ cell bodies were largely absent (Fig. 2C). Quantitative analysis confirmed significant regional differences, indicating that the CeA and BNST contained substantially more CRF⁺ neurons than the dorsal striatum (Fig. 2D; CeA versus DS, Q = 6.62, *p < 0.05; BNST versus DS, Q = 4.54, *p < 0.05). Notably, the CeA exhibited a similar density of CRF⁺ neurons to the BNST (Fig. 2D; Q = 1.40, p > 0.05). The apparent lack of CRF⁺ cell bodies suggests that CRF signaling in the dorsal striatum originates from CRF⁺ neurons in the CeA and BNST.

Figure 2.

Dorsal Striatum CINs Receive Monosynaptic CRF⁺ Inputs from the CeA and BNST

To test whether CRF⁺ neurons in the CeA and BNST project to the dorsal striatum, we infused a retrograde adeno-associated virus into the dorsal striatum of CRF-Cre;tdTomato rats. This strategy labels CRF⁺ neurons (tdTomato⁺) that project to the dorsal striatum with GFP (Fig. 3A). Confocal imaging revealed GFP⁺ neurons in both the BNST (Fig. 3B) and CeA (Fig. 3C), with substantial overlap between GFP-labeled dorsal striatum-projecting neurons and tdTomato-positive (CRF⁺) cells in both regions. This indicates that CRF⁺ neurons from the CeA and BNST project to the dorsal striatum. Despite a similar overall CRF⁺ neuron density in the CeA and BNST (Fig. 3D; t₁₅ = 1.06, p > 0.05), the proportion of dorsal striatum-projecting CRF⁺ neurons was significantly higher in the BNST than in the CeA (Fig. 3E; t₁₅ = -5.01, ***p < 0.001). The density of dorsal striatum-projecting CRF⁺ neurons was also higher in the BNST than the CeA (Fig. 3F; t₁₅ = -3.07, **p < 0.01). These results indicate that CRF⁺ neurons in the CeA and BNST project to the dorsal striatum.

Figure 3.

To test whether CRF⁺ neurons in the CeA and BNST make monosynaptic projections to dorsal striatal CINs, we performed opsin-assisted functional circuit tracing. Given that both the CeA and BNST are primarily GABAergic nuclei [58, 59], we examined whether optogenetic stimulation of CRF-containing projections to the dorsal striatum induces inhibitory monosynaptic currents in CINs. To test this, we infused AAV-FLEX-Chrimson-tdTomato into the CeA and BNST of CRF-Cre;ChAT-eGFP mice so that we could selectively stimulate CRF-containing fibers in the dorsal striatum while recording from GFP-labeled CINs (Fig. 3G). We found that optogenetic stimulation of CRF⁺ fibers with yellow light evoked fast synaptic currents (within ∼6.5 ms from the stimulation onset) in dorsal striatal CINs (Fig. 3H-3J). This response was abolished by TTX (Fig. 3H, 3I; X²(3) = 21.9, p < 0.001; BL versus TTX: z = 3.10, *p < 0.05) but restored using TTX+4-AP (Fig. 3H, 3I; TTX vs TTX+4-AP: z = 4.26, ***p < 0.001), suggesting the existence of monosynaptic transmission. Notably, the restored synaptic response was predominantly blocked by the GABA_A receptor antagonist bicuculline (Fig. 3H, 3I; TTX+4-AP versus TTX+4-AP+Bic: z = 3.10, *p < 0.05), demonstrating that evoked currents are GABAergic. Together, these results demonstrate that dorsal striatal CINs receive monosynaptic GABAergic inputs from CRF⁺ neurons in the CeA and BNST, suggesting that these neurons project to the dorsal striatum and may release CRF to modulate cholinergic signaling.

Striatal CINs Express CRFR1

Having established that CINs receive monosynaptic inputs from CRF⁺ neurons in the CeA and BNST, we next sought to confirm that CINs express the CRF receptor CRFR1 [15, 60, 61]. To address this, we used CRFR1-Cre-tdTomato rats [28], in which CRFR1-expressing neurons express Cre recombinase and are visualized via tdTomato fluorescence. Confocal imaging of striatal sections from these rats confirmed that CRFR1-expressing neurons are distributed throughout the striatum (Fig. 4A). To verify that CRFR1-positive (CRFR1⁺) neurons are also cholinergic, we stained sections with an anti-ChAT antibody and acquired confocal images (Fig. 4A). We found that ∼25% of rat CINs express CRFR1 (Fig. 4B, 4C; Mann-Whitney U = 0.00, ***p < 0.001). To determine whether this expression pattern is conserved across species, we examined CRFR1-GFP mice and performed ChAT immunostaining on striatal sections (Fig. 4D). We found that approximately 10% of CINs expressed CRFR1 (Fig. 4E), indicating that while the proportion is lower than in rats, a subset of CINs in both species express CRFR1. These findings align with previous reports showing that a subset of striatal CINs express CRFR1, supporting a role for CRFR1-mediated signaling in the striatum.

Figure 4.

CRF Enhances CIN Activity and Acetylcholine Release in the Dorsal Striatum

Following the finding that CRF⁺ neurons send inputs to the dorsal striatum, we next investigated how CRF influences spontaneous CIN firing activity. Cell-attached recordings of GFP⁺ neurons in the dorsal striatum were conducted in ChAT-eGFP mice before and during bath application of CRF (100 nM) (Fig. 5A). We found that CRF significantly increased CIN firing frequency (Fig. 5B, 5C; t₍₈₎ = -24.08, ***p < 0.001). This effect was abolished by pretreatment with the CRFR1 antagonist NBI 35695 (Fig. 5C; Mann-Whitney U = 0.00, ***p < 0.001), confirming that CRF potentiates CIN firing via CRFR1 activation in the dorsal striatum.

Figure 5.

Because CINs are the primary source of ACh in the dorsal striatum, we next examined whether CRF-induced CIN activation modulates ACh release. To do so, we infused an adeno-associated virus (AAV) introducing a genetically encoded ACh sensor (AAV-hSyn-GRAB_ACh4m) into the dorsal striatum of wildtype mice [40–45]. Live-tissue confocal imaging was performed on brain slices containing the dorsal striatum 14 days post-infusion to monitor ACh levels (Fig. 5D). Bath application of CRF produced a robust ACh fluorescence signal (Fig. 5E, 5F; Mann-Whitney U = 7.00, *p < 0.05), indicating that CRF enhances ACh release. These results suggest that CRF-driven CIN activation increases ACh release in the dorsal striatum.

To further determine whether direct activation of CRF-expressing fibers modulates CIN activity, we used an optogenetic approach. We generated CRF-Cre;Ai32;ChAT-eGFP mice to selectively stimulate CRF⁺ terminals in the dorsal striatum. Cell-attached recordings were conducted in CINs while blue light (470 nm, 50 Hz, 60 s) activated local ChR2-expressing CRF⁺ fibers (Fig. 5G). Burst stimulation of CRF⁺ terminals significantly and reversibly increased CIN firing in the presence of DNQX and bicuculline (Fig. 5H, 5I; pre vs. stim, q = 3.69, *p < 0.05; stim vs. post, q = 11.82, ***p < 0.001). However, application of a CRFR1 antagonist abolished this effect (Fig. 5J, 5K; F_(2,16) = 0.96, p = 0.41), confirming that the increase in CIN firing was mediated by CRFR1 signaling. Together, these findings demonstrate that CRF enhances CIN activity and ACh release in the dorsal striatum.

Acute Alcohol Application Attenuates CRF-Mediated Enhancement of CIN Firing

Given that acute alcohol exposure suppresses CIN activity [20], we next examined how acute alcohol application and subsequent withdrawal influence CRF-mediated enhancement of CIN firing. We first assessed the effect of acute alcohol by measuring spontaneous CIN activity before, during, and after 50 mM alcohol application in dorsal striatal slices from ChAT-eGFP mice. Consistent with previous reports [20], acute alcohol significantly suppressed CIN spontaneous firing, an effect reversed after a washout period, which we refer to here as withdrawal (Fig. 6A, 6B; Mann-Whitney U = 0.00, *p < 0.05).

Figure 6.

To determine how prior alcohol exposure (modeling early withdrawal) affects CRF-induced enhancement of CIN firing, we pretreated dorsal striatal slices with alcohol (50 mM) for 1 h outside of the recording chamber (Fig. 6C), followed by CRF application during recordings. In untreated control slices, CRF significantly increased spontaneous CIN firing, consistent with our previous findings (Fig. 6D; BL versus CRF, q = 11.51, ***p < 0.001; see also Fig. 5B). When alcohol was subsequently applied after CRF, firing decreased but remained elevated relative to baseline (Fig. 6D; CRF versus EtOH, q = 6.21, **p < 0.01; BL versus EtOH, q = 5.29, **p < 0.01), likely due to a residual CRF effect (Fig. 5B).

In slices pretreated with alcohol (mimicking early withdrawal), CRF still increased CIN firing, but subsequent alcohol application did not fully return firing to baseline levels (Fig. 6E; BL versus CRF, q = 5.20, **p < 0.01; CRF versus EtOH, q = 6.21, p = 0.05). This suggests that alcohol pretreatment (withdrawal state) attenuates CRF-induced CIN activation and diminishes alcohol’s subsequent suppressive effects. Notably, when comparing the change in firing from baseline to CRF application, alcohol pretreatment blunted CRF-induced CIN activation (Fig. 6F; t₍₁₈₎ = 2.70, *p < 0.05). Together, these findings indicate that both acute alcohol exposure and early withdrawal attenuate CRF-mediated enhancement of CIN firing, supporting the conclusion that alcohol modulates striatal cholinergic signaling.

Discussion

This study identifies a circuit in which CRF-positive neurons in the CeA and BNST provide direct input to dorsal striatal CINs that express CRFR1. CRF enhances CIN excitability and ACh release, linking CRF to cholinergic modulation. In addition, alcohol exposure and withdrawal blunt this effect, suggesting that CRF signaling to dorsal striatal CINs may contribute to mechanisms relevant to alcohol use disorder. Using monosynaptic and retrograde circuit tracing, we identified direct projections from CRF-expressing neurons in the CeA and BNST to dorsal striatal CINs. Functional electrophysiology shows that CRF enhances CIN excitability and promotes ACh release via CRFR1 receptors. However, acute alcohol exposure and withdrawal disrupted this excitatory effect, indicating that alcohol interferes with CRF-dependent cholinergic modulation. These findings identify a CRF–CIN circuit that is sensitive to alcohol-induced modulation. Striatal cholinergic signaling is essential for behavioral flexibility [62], and alcohol exposure is known to impair CIN activity and ACh release [49], leading to reduced flexibility. Thus, alcohol-induced alterations in CRF–CIN signaling may disrupt cholinergic control of striatal circuits, providing a potential mechanism by which alcohol promotes cognitive rigidity and compulsive reward-seeking behaviors characteristic of AUD.

Monosynaptic Inputs from the CeA and BNST to CINs

Tracing experiments reported here show that dorsal striatal CINs receive direct synaptic input from CRF-expressing neurons in the CeA and BNST, regions involved in stress and emotion [58, 63]. While previous studies have identified CeA and BNST projections to the striatum, they did not show that they arise from CRF neurons or that they target CINs [52, 64–66]. Our results support the presence of a direct CRF-to-CIN pathway, linking CRF release to cholinergic modulation. Given the role of CINs in striatal output, response flexibility, and reward learning [50, 67–70], this circuit likely contributes to stress-driven behavioral adaptations [58, 71–74]. A limitation of our optogenetic approach is the use of CRF-Cre;Ai32 mice, in which ChR2 is expressed in all CRF+ neurons. Thus, although our tracing confirmed CeA and BNST inputs, we cannot exclude contributions from other CRF+ populations. Future work using projection-specific targeting approaches will be required to isolate CeA and BNST contributions.

Tracing and receptor expression studies were performed in mice and rats in a largely non-overlapping manner. While mice and rats share many conserved amygdalostriatal components, we did not attempt direct cross-species comparisons, and our findings should therefore be interpreted as species-specific. In rats, CRFR1 expression was largely restricted to a subset of CINs, consistent with previous reports. In mice, the histological distribution of CRFR1 remains less well defined; however, our recordings from GFP-labeled CINs in ChAT-GFP mice demonstrated that bath-applied CRF increased CIN firing in a CRFR1-dependent manner. These recordings were conducted in the presence of glutamatergic and GABAergic antagonists, and in some cases TTX, ensuring that CINs were functionally isolated from upstream inputs. Because striatal acetylcholine arises almost exclusively from CINs, with only a minor contribution from brainstem cholinergic afferents, it is most likely that CRF-induced increases in acetylcholine reflect direct CRFR1 signaling in CINs. Nevertheless, the possibility that CRFR1 is expressed in other striatal cell types remains an important open question. Another unresolved question is whether CRFR1+ CINs are equally abundant across striatal subregions. While representative images may appear to suggest higher CRFR1⁺ CIN density in the DLS compared to the DMS, we did not quantify this systematically. Future work will be needed to determine whether such regional differences exist, as they could have important implications for dorsal striatal function.

Although CRFR1 expression has been reported in a subset of striatal CINs, we found that the majority of mouse CINs increased firing in response to bath-applied CRF. Using a CRFR1-GFP reporter mouse, only ∼10% of CINs showed detectable somatic CRFR1-GFP expression, indicating that widespread CIN responsiveness cannot be explained solely by direct receptor expression at the soma. Reporter lines may underestimate functional CRFR1 expression, particularly if receptors are expressed at low levels or localized to distal neuronal compartments. Consistent with a CRFR1-dependent mechanism, the CRF-induced increase in CIN firing was abolished by a selective CRFR1 antagonist. In addition, CRF may act indirectly by modulating presynaptic inputs to CINs, and electrical coupling among CINs may allow excitation of a subset of neurons to propagate across the network [75]. Together, these mechanisms provide a parsimonious explanation for broad CIN sensitivity to CRF despite limited reporter-detected CRFR1 expression.

We found no CRF⁺ neurons in the dorsal striatum, indicating that CRF signaling in this region originates from extrinsic inputs, including the CeA and BNST. However, CRF+ neurons have been reported in the ventral striatum, where they play a role in learning-related processes [76]. Thus, CRF’s contribution to striatal function may differ along the dorsal-ventral axis, with intrinsic ventral striatal CRF⁺ neurons complementing extrinsic dorsal striatal CRF inputs. These sources of CRF may modulate striatal circuits involved in selecting behaviors flexibly, a process critical for adaptive responses to stress. Electrophysiological experiments further show that CRF fiber stimulation enhances CIN excitability even in the presence of synaptic blockers, supporting a direct modulatory role for CRF signaling on cholinergic tone in the striatum. Moreover, the transient effects of endogenous CRF release, compared to the prolonged activation from CRF bath application, suggest that CRF signaling may generate temporally dynamic patterns of CIN activity to influence behavioral output.

Alcohol Disrupts CRF-Mediated CIN Excitation

One key observation is that alcohol exposure disrupts CRF-mediated CIN excitation. Both acute alcohol exposure and withdrawal attenuate the CRF-induced increase in CIN activity, indicating that alcohol reduces CIN responsiveness to CRF, a stress-related neuromodulator. Because stress is a well-established trigger for alcohol relapse [77–79], the observed alcohol-induced disruption of CRF signaling may impair the brain’s ability to adaptively respond to stress, thereby increasing susceptibility to relapse. Such dysregulation may compromise stress resilience mechanisms normally mediated by CRF–cholinergic interactions within the striatum and related corticostriatal circuits. Under normal stressful conditions, CRF release onto CINs can modulate cholinergic signaling to adjust behavioral strategies and promote flexible adaptation to environmental demands. However, alcohol-induced impairment of CRF–CIN communication and CIN responsiveness may blunt this adaptive flexibility [49, 62]. Consequently, stress may instead bias behavior toward habitual or compulsive alcohol seeking, leading to relapse. These findings highlight a potential pathway through which chronic alcohol exposure erodes adaptive stress coping and promotes relapse vulnerability. While these alcohol effects were modest in magnitude, they were consistent across recordings and statistically reliable. It is important to note, however, that ex vivo slice physiology may underestimate ethanol’s impact due to factors such as washout, diffusion barriers, and the absence of an intact network state. Thus, future in vivo studies are needed to evaluate the robustness and behavioral relevance of this modulation. It also remains to be determined whether ethanol pre-exposure alters subsequent CIN responses to ethanol in the absence of CRF, an important question for future studies.

One possibility is that alcohol exposure downregulates CRFR1 expression [80, 81] or alters CIN function [49], reducing their responsiveness to stress-related input [82, 83]. Another is that alcohol may interfere with intracellular signaling cascades downstream of CRFR1 activation, leading to diminished excitability and ACh release. Another possibility is that alcohol and CRF may converge on CINs through an occlusion mechanism, in which prior activation of CRFR1 reduces the efficacy of subsequent CRF signaling. Although our ex vivo preparation does not readily permit a direct test of this hypothesis, future in vivo approaches with high temporal control will be important to evaluate this possibility.

Implications for Alcohol Use Disorder

By delineating a CRF-to-CIN pathway linking the extended amygdala to the dorsal striatum, our findings provide insight into how stress and alcohol may interact at the circuit level to influence striatal processing relevant to behavior. Given the role of CINs in habit learning and behavioral flexibility [84], disruption of this stress-sensitive cholinergic circuit may underlie the decision-making deficits and compulsive alcohol-seeking behavior observed in AUD [48–50, 85–87].

In summary, we identify a direct CRF-positive projection from the CeA and BNST to dorsal striatal CINs, revealing a new mechanism by which stress can modulate cholinergic signaling in the striatum. We show that CRF enhances CIN excitability and ACh release, and that alcohol exposure attenuates this modulation. These findings highlight the CRF-CIN circuit as a potential site of vulnerability in AUD and suggest that restoring CRF signaling may help counteract stress and alcohol-induced striatal dysfunction. Future work should assess whether pharmacological or circuit-based interventions targeting CRFR1 or CINs can mitigate behavioral impairments in individuals with AUD.

Data availability

All figures contain the numerical data points used to generate the figures.

Acknowledgements

We thank Yufei Huang for discussions and supplementary data. This research was supported by NIAAA R01AA021505 (JW), R01AA027768 (JW), U01AA025932 (JW), R01MH112768 (NJ), and R21AG086907 (NJ).

The authors declare no competing financial interests.

Additional information

Funding

HHS | NIH | National Institute on Alcohol Abuse and Alcoholism (NIAAA) (R01AA021505)

• Jun Wang

HHS | NIH | National Institute on Alcohol Abuse and Alcoholism (NIAAA) (R01AA027768)

• Jun Wang

HHS | NIH | National Institute on Alcohol Abuse and Alcoholism (NIAAA) (U01AA025932)

• Jun Wang

HHS | NIH | National Institute of Mental Health (NIMH) (R01MH112768)

• Nicholas J Justice

HHS | NIH | National Institute of Mental Health (NIMH) (R21AG086907)

• Nicholas J Justice