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. Author manuscript; available in PMC: 2025 Nov 1.
Published in final edited form as: Alcohol. 2024 Jun 17;120:1–14. doi: 10.1016/j.alcohol.2024.06.001

Dysregulation of Neurotrophin Expression in Prefrontal Cortex and Nucleus Basalis Magnocellularis During and After Adolescent Intermittent Ethanol Exposure

Brian T Kipp 1, Polliana T Nunes 1, Lisa M Savage 1
PMCID: PMC11390331  NIHMSID: NIHMS2001864  PMID: 38897258

Abstract

A preclinical model of human adolescent binge drinking, adolescent intermittent ethanol exposure (AIE) recreates the heavy binge withdrawal consummatory patterns of adolescents and has identified the loss of basal forebrain cholinergic neurons as a pathological hallmark of this model. Cholinergic neurons of the nucleus basalis magnocellularis (NbM) that innervate the prefrontal cortex (PFC) are particularly vulnerable to alcohol related neurodegeneration. Target derived neurotrophins (nerve growth factor [NGF] and brain-derived neurotrophic factor [BDNF]) regulate cholinergic phenotype expression and survival. Evidence from other disease models implicates the role of immature neurotrophin, or proneurotrophins, activity at neurotrophic receptors in promoting cholinergic degeneration; however, it has yet to be explored in adolescent binge drinking. We sought to characterize the pro- and mature neurotrophin expression, alongside their cognate receptors and cholinergic markers in an AIE model. Male and female Sprague Dawley rats underwent 5g/kg 20% EtOH or water gavage on two-day-on, two-day-off cycles from post-natal day 25–57. Rats were sacrificed 2 hours, 24 hours, or 3 weeks following the last gavage, and tissue were collected for protein measurement. Western blot analyses revealed that ethanol intoxication reduced the expression of BDNF and vesicular acetylcholine transporter (vAChT) in the PFC, while NGF was lower in the NbM of AIE treated animals. During acute alcohol withdrawal, proNGF in the PFC was increased while proBDNF decreased, and in the NbM proBDNF increased while NGF decreased. During AIE abstinence, the expression of neurotrophins, their receptors, and vAChT did not differ from controls in the PFC. In contrast, in the NbM the expression of both NGF and choline acetyltransferase (ChAT) were reduced long-term following AIE. Taken together these findings suggest that AIE alters the expression of proneurotrophins and neurotrophins during intoxication and withdrawal that favor prodegenerative mechanisms by increasing the expression of proNGF and proBDNF, while also reducing NGF and BDNF.

Keywords: Adolescent, Ethanol, Withdrawal, Abstinence, Neurotrophins

Introduction

Adolescence is an evolutionarily conserved period of development across species, where cortical forebrain regions undergo maturational changes that parallel further refinement of prefrontal cortical dependent executive processes. This period of development in humans often coincides with initiation of alcohol consumption (Crews et al., 2019; Spear, 2016). Binge ethanol consumption during adolescence has been shown to not only increase the susceptibility to substance use disorders in adulthood, but also causes reduced prefrontal cortical connectivity and volume (De Bellis et al., 2005). The AIE model mimics the episodic pattern of binge drinking observed in adolescents and early adulthood which consistently produces long-lasting impairments in cognition, and brain function (Crews et al., 2019; Nunes et al., 2019; Spear, 2016). Cholinergic neurons of the basal forebrain appear to be particularly sensitive to the effects of adolescent, but not adult, ethanol exposure as a global reduction in ChAT expression is observed following AIE, corresponding with reductions in acetylcholine (ACh) release in the PFC and orbitofrontal cortex (OFC, Crews et al., 2021; Fernandez & Savage, 2017; Kipp et al., 2021; Vetreno et al., 2014; Vetreno & Crews, 2018).

Cholinergic neurons are dependent on target-derived neurotrophins for maintenance of phenotype and cholinergic gene transcription, and changes in neurotrophin expression have been observed to induce pathological dysfunction across multiple disease states (Allard et al., 2018; Allard et al., 2012; Boskovic et al., 2019; Fahnestock & Shekari, 2019). Whereas NGF, and particularly BDNF, have been studied in relation to prenatal and adult alcohol exposure and alcohol use disorder (AUD), there is a considerable gap in the literature regarding alcohol-related changes in neurotrophin expression in adolescent models of AUD (Davis, 2008). We have previously observed a significant decrease in the expression of mature BDNF in the frontal cortex 1-hour following the last ethanol gavage in adolescent and adult-treated male Sprague Dawley rats (Fernandez et al., 2017). However, 24-hours following the last ethanol exposure, the blunting of mature BDNF persisted in early adolescent treated rats only, and the expression of mature BDNF in adult ethanol treated rats rebounded to levels much higher than controls. This rebounding of BDNF expression in adult animals during acute ethanol withdrawal may facilitate recovery from ethanol toxicity, as neurotrophins has been shown to be neuroprotective (Knüsel et al., 1992; Moser et al., 2006; Niewiadomska et al., 2002; Péan et al., 2000; Widmer et al., 1993; Zassler & Humpel, 2006). Moreover, voluntary exercise, which increases the release of neurotrophins (Hall et al., 2018; Hall & Savage, 2016), has been shown to both protect and recover losses in basal forebrain cholinergic phenotype expression following AIE (Vetreno et al., 2020; Vetreno & Crews, 2018). The high affinity NGF receptor, tropomyosin receptor kinase A (TrkA), is also a critical regulator of cholinergic phenotype expression, and reductions in NGF/TrkA binding leads to cholinergic neuronal atrophy and reductions in cholinergic marker expression (Gage et al., 1988; Lazo et al., 2010).

Immature forms of NGF and BDNF, or proNGF and proBDNF, are biologically active, interact with the pan neurotrophin receptor, p75NTR, that can lead to the engagement of prodegenerative mechanisms. While their role in mediating neuronal pathology is most closely examined in Alzheimer’s Disease, activity at the p75NTR has been shown to participate in a myriad of disease states including traumatic brain injury, cerebrovascular diseases, opioid withdrawal, Huntington’s Disease, and aging (Dasgupta et al., 2023; Fahnestock & Shekari, 2019; Suelves et al., 2019; Xiong et al., 2022; Xu et al., 2019). The expression of proneurotrophins hasn’t been widely explored in developmental models, let alone developmental models of ethanol exposure. It has been suggested for some time that the dysregulation of neurotrophic activity may be involved in ethanol-induced neurodegeneration, including brain damage due to developmental ethanol exposure (Davis, 2008). However, changes in the neurotrophic response to toxic developmental levels of ethanol are likely to be time and regional specific (Davis, 2008). In adults, however, 6 weeks of 10% (w/v) ethanol in drinking water of male C57BL6 mice increased proBDNF expression in the PFC, midbrain, and amygdala; but ethanol had no effect on mature BDNF expression during intoxication (Popova et al., 2020). Additionally, prohormone convertase 1/3, which facilitates the maturational cleavage of pro- sequences from proneurotrophins to form mature neurotrophins is reduced following 16 days of 7% ethanol treatment in adult male rats, with a significant correlation between the amount of voluntary ethanol consumption and the degree of prohormone convertase 1/3 reduction (Navarro et al., 2013).

Thus, a growing body of literature implicating proneurotrophins in mediating neurodegeneration across multiple disease states begs the question as to whether proneurotrophin mediated degeneration is truly disease state-specific, or more likely, a conserved mechanism of degeneration that also extends to ethanol-mediated neurodegeneration. The findings point to the possibility that although the inciting incident in these various disease states may differ, the activity at the p75NTR may be a convergence point in neurodegeneration.

This study sought to replicate and expand upon our previous findings (Fernandez et al., 2017) by measuring changes in both pro-, and mature neurotrophins, as well as their associated receptors during intoxication, withdrawal, and protracted withdrawal. By examining ethanol-induced changes in neurotrophin and proneurotrophin responses in intoxication, withdrawal, and abstinence in sites of cholinergic nuclei (NbM) and terminal projection regions (PFC), we can characterize fluctuations and their association with cholinergic markers.

Materials and Methods

1.1. Subjects

A total of 50 male and 53 female Sprague Dawley rats, bred at Binghamton University, from breeders sourced from Envigo (Indianapolis, IN) were used for this study. Approximately 9 rats per sex, were randomly assigned to experimental conditions. Only one rat per sex was assigned to an experimental condition from a given litter. The rats were reared in a vivarium at Binghamton University under a 12-hour light-dark cycle (0700 – 1900), where temperature and humidity were controlled. Throughout the experiment, the rats were given unrestricted access to food chow (Purina LabDiet 5012) and tap water. Additionally, nesting papers and wooden chew blocks were provided as environmental enrichment. The Institutional Animal Care and Use Committee (IACUC) at Binghamton University approved all experimental procedures.

1.2. Ethanol Treatment

The rats were treated with 5g/kg of 20% ethanol (AIE) through intragastric gavage once a day following a 2-day-on/2-day-off schedule spanning postnatal day (PND) 25–57 (see Figure 1A; Vetreno et al, 2020). Control (CON) animals underwent the same treatment schedule but received an equivalent volume of tap water. One hour following the 8th gavage (PND 38), tail blood samples were collected from AIE treated animals, and blood ethanol concentrations (BEC) were measured (GM7 Analyzer, Analox, London, UK). This protocol consistently yields BECs between 180–250 mg/dl in AIE-treated animals. Three time points of AIE treatment were assessed for changes in protein expression. One cohort was euthanized 2-hrs following the last gavage, another cohort was euthanized 24-hours following the last gavage (PD 58), and the final cohort was euthanized 3 weeks following the last gavage (PD78).

Figure 1: Experiment 1 Treatment Timeline, BEC’s, and Growth Curves.

Figure 1:

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Schematic demonstrating treatment timeline for Experiment 1. Male and female Sprague Dawley rats were treated with either 5g/kg EtOH or volume matched amount of water on a two-day-on two-day-off schedule from post-natal day 25–57. Subjects were euthanized and tissue collected on either PND57 (2 hours after the last gavage), PND 58 (24 hours after the last gavage), or PND78 (3 weeks following the last gavage) (A). One hour following the 8th gavage, tail bloods were collected from AIE treated animals BEC’s were measured (B). Change in body weight over the course of AIE treatment was recorded in males (C) and females (D). Depiction of tissues punched for western analysis, Image from Paxinos and Watson (2014) (E-F). Graphs depict group mean ± SEM. ** indicates p < 0.01; **** indicates a p < 0.001. Final N: CON males (n = 26), CON females (n = 27), AIE males (n = 25), AIE females (n = 26)

1.3. Tissue Preparation

Rodents were briefly restrained and sacrificed via rapid decapitation (Decapicones, ThermoFisher Scientific, Waltham, MA, USA) and flash frozen in 2-methylbutane (Sigma Aldrich, St. Louis MO) before storage at −80°C. Brains were mounted on a Cryostat (CM1510, Leica Microsystems, Wetzlar, Germany) and NbM and PFC tissue were micropunched (1.0–2.0mm EMS-Core Sampling Tools, Electron Microscopy Sciences, Hatfield, PA, USA) at −20 °C and stored at −80 °C prior to lysis (Paxinos & Watson, 2006). Tissues were homogenized in lysis buffer (1% SDS, 1mM EDTA, 10mM Tris) in the presence of protease inhibitors (Halt™ Protease Inhibitor Cocktail, Thermo Scientific, Waltham, MA, USA) and centrifuged at 4°C, 12,000g for 30 minutes. Lasty, protein concentrations in tissue homogenate were determined using a bicinchoninic acid method (Pierce, Rockford, IL, USA) and compared to bovine serum albumin standards.

1.4. Western Blotting

Total protein samples (30 μg) of the PFC and NbM samples were denatured and separated by electrophoresis on Novex™ 8–16% Tris-Glycine sodium dodecyl sulfate polyacrylamide gels (Invitrogen, Carlsbad, CA, USA), transferred to a polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA, USA). To reduce potential antibody cross reactivity, and to eliminate the need for a stripping buffer, blots were cut according to the following KDa markers of the colorimetric ladder – 35kDA, 50kDA, 100kDA, and 140 kDA, producing 4 narrow range blots (Spectra™ Multicolor Broad Range Protein Ladder, Thermo Scientific, Rockford, IL, USA). Blots were then blocked for one hour in 5% BSA, 0.01% Tween-20 in TBS before incubating overnight in the block solution with primary antibodies (see Table 1 - NGF, BDNF, TrkA, TrkB, p75NTR, ChAT, vAChT, ®-Actin). The following day, blots were briefly washed in TBSt before 1-hr incubation in secondary antibodies (see Table 1), conjugated with Horse Radish Peroxidase. Signal was generated with ECL Western Blotting Detection Reagents (Thermo Scientific, Rockford, IL, USA) digitally captured (Azure 600, Azure Biosystems, Dublin, CA, USA), and measured (Azure Spot Pro, Azure Biosystems, Dublin, CA, USA). First, the background was subtracted using a 3-pixel rolling ball radius prior to signal intensity measurement via area under the curve measurements. Band intensity was then transformed to relative measurement compared to ®-Actin intensity. When doublets in bands could not be resolve one from the other, they were analyzed together. Data was then transformed to represent the expression of the target protein relative to protein expression in control animals, expressed as percent change from controls.

Table 1 –

Western Blot Antibody Information

Protein Primary Antibody Origin Primary Concentration Secondary Antibody Origin Secondary Concentration
NGF Abcam, ab52918 Rabbit 1:150 Abcam, ab205718 Goat 1:250
proNGF Abcam, ab52918 Rabbit 1:150 Abcam, ab205718 Goat 1:250
BDNF Abcam, ab108319 Rabbit 1:200 Abcam, ab205718 Goat 1:500
proBDNF Abcam, ab108319 Rabbit 1:200 Abcam, ab205718 Goat 1:500
P75NTR EMD Millipore, 07–476 Rabbit 1:300 Abcam, ab205718 Goat 1:500
ChAT EMD Millipore, AB144P Goat 1:200 Abcam, ab6885 Donkey 1:1000
vAChT EMD Millipore, ABN100 Goat 1:600 Abcam, ab6885 Donkey 1:1000
TrkA Abcam, ab302524 Rabbit 1:1000 Abcam, ab205718 Goat 1:2000
TrkB Abcam, ab187041 Rabbit 1:1000 Abcam, ab205718 Goat 1:2000
®-Actin Origene, TA349013 Chicken 1:1000 Abcam, ab97135 Goat 1:2000
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1.5. Statistical Analyses

Changes in body weight during treatment were analyzed with a repeated measures ANOVA with between-subject factors of Exposure (CON and AIE), and a within-subject factor of treatment day. Due to the differences in weight trajectories during adolescence, males and females were analyzed separately for change in weight gain over the course of treatment. However, for all other analyses males and females were analyzed together and the between-subject factor of Sex was included. For BEC data, a one-way ANOVA (Sex) was used to compare BECs of male and female AIE treated rats. Three different 2-way ANOVAs (Exposure, Sex) were used to compare protein expression at the 2-hour, 24-hour, and 3-week timepoints. For the western blotting data set a Grubb’s test was utilized to ensure the robustness of data prior to group comparison, final group n’s are displayed in the figure captions, and raw western blot strips are shown in supplemental materials (Supplemental Figures 4–9). When an exposure effect was observed in the absence of sex differences, the ANOVA was collapsed across that factor, but data is uniquely marked as a function of sex. To better map the relationships in protein expression, Pearson’s correlation matrices were conducted between the mPFC and NbM, resulting in 107 contrasts per group and timepoint. To control for the number of contrasts, significance thresholding was adjusted based on the Benjamini-Hochberg method for false discovery rate. Since the adjusted p-value for significance using the Benjamini-Hochberg method was determined to be p = 0.0028, p values less than 0.0028 were deemed significant with a false discovery rate of 0.05. All statistical analyses for this study were conducted using IBM SPSS version 25.

Results

2.1. AIE Treatment –BECs and Growth Curves

On the 8th day of gavage, tail bloods were collected for BEC measurement. Males (211.0 mg/dl; SEM = 10.01) and females (201.6 mg/dl; SEM = 8.12) did not differ significantly in BECs (p = 0.46), as shown in Figure 1B. Similarly, the between subjects ANOVA did not detect significant differences between the three time point conditions in BEC levels (p = 0.98). The level of BEC in this model replicates extreme binge alcohol levels in adolescents and emerging adults (0.23 BAC; Hua et al., 2020).

Over the course of AIE treatment the body weights of both water and ethanol-treated males increased from the beginning to end of treatment (F (2.175, 95.71) = 8334, p < 0.0001; ηp2 = 0.995; See Figure 1C). Moreover, a significant main effect of Exposure was also detected (F(1,44) = 15.438, p < 0.001; ηp2 = 0.260: AIE-treated males had lower body weights during treatment compared to CON males. Similarly, the body weights of females also increased over the course of AIE treatment (F(2.73, 112.08) = 3959.84, p < 0.0001; ηp2 = 0.990), and AIE females had lower body weights compared to CON females (F(1,45) = 7.310, p < 0.05; ηp2 = 0.140; See Figure 1D). No significant effects of Time point were detected in the body weights of male or female rats (Males: p = 0.38; Females: p = 0.55).

2.2. Intoxication: 2-Hour Time point

A. Prefrontal Cortex

As shown in Figure 2, the relative expression of proNGF and NGF in AIE-treated rats compared to controls did not differ as across Exposure conditions (Pro-NGF: p = 0.47; NGF: p = 0.087), or Sex (ProNGF: p = 0.085; NGF: p = 0.45), or their interaction (proNGF; p = 0.94; NGF (p = 0.42). However, CON and AIE treated rats differed in mature BDNF expression (F(1,28) = 6.619, p = 0.016; ηp2=0.191), but no sex differences or an Exposure X Sex interactions were found (both p’s> 0.43). Two-hours following the last gavage, BDNF expression in AIE treated rats was lower in the PFC relative to the CON condition. In contrast, proBDNF expression was not found to be affected as a function of Exposure (p = 0.19), Sex (p = 0.29), or an Exposure X Sex interaction (p = 0.57). The cholinergic marker vAChT was also found to be affected by Exposure (F(1,28) = 5.541, p = 0.026; ηp2= 0.165), without a main effect of, or interaction with, Sex (both p’s> 0.65). AIE-treated rats had a reduction in vAChT expression compared to CON rats. However, the expression of TrkA, TrkB, nor p75NTR significantly differed as a function of Exposure, Sex, or their interaction (all p’s>0.18).

Figure 2: Prefrontal Cortex – 2-hours post AIE.

Figure 2:

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Neurotrophin and cholinergic marker expression 2-hours following the last gavage measured with western blot and ®-actin housekeeper. Data represents the relative protein expression compared to controls. proNGF, NGF, and proBDNF expression did not differ between AIE and controls (A-C); However, BDNF and vAChT expression were significantly reduced in AIE treated animals (D). Neurotrophin and proneurotrophin receptors TrkA, TrkB, and p75NTR protein expression did not change as a result of AIE exposure (E-H). Blot representations are in I. Open circles represent males, closed circles represent females. Graphs depict group mean ± SEM. * indicates a p < 0.05.

Final N: CON males (n = 7–8), CON females (n = 8), AIE males (n = 9), AIE females (n = 7).

Outlier removed: proNGF (n=1 CON male).

B. Nucleus Basalis Magnocellularis

Significant differences were found between CON and AIE-treated rats in mature NGF expression (F(1,28) = 9.708, p = 0.004; ηp2= 0.257; see Figure 3B), where AIE treatment reduced NGF expression in the NbM compared to CON. Additionally, NGF expression also significantly differed across Sex (F(1,28) = 6.824, p = 0.014, ηp2= 0.196), where NGF expression in males was lower than females. No significant effects of Exposure or Sex were detected of pro-NGF expression (Figure 3A). There was a significant Exposure X Sex interaction (F(1,28) = 4.989, p = 0.034; ηp2= 0.151; Figure 3C) of proBDNF expression in the NbM. Fisher’s LSD comparisons did not reveal any significant differences; however, CON males (89.11%, SEM = 4.94%) trended towards having lower proBDNF expression compared to AIE males (118.27%, SEM = 13.36%) (p = 0.0572). No differences were detected as a function of Exposure or Sex, or their interactions, with regards to BDNF, ChAT, TrkA, TrkB, or p75NTR expression (all p’s >0.09; see Figure 3D–I).

Figure 3: Nucleus Basalis Magnocellularis – 2 hours post AIE.

Figure 3:

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NbM protein expression of pro and mature neurotrophins, their receptors, and cholinergic marker ChAT 2 hours following the last ethanol gavage measured with western blot and b-actin housekeeper. Data represents mean relative protein expression compared to CON, ± SEM. Expression of proNGF did not differ between CON and AIE treated animals (A). In contrast, there was a main effect of Exposure on mNGF expression: AIE significantly lowered mNGF in females, alongside a reduction in males (B). There was an Exposure X Sex interaction for proBDNF expression: AIE males had greater proBDNF expression than CON males and AIE females (C), BDNF, ChAT, TrkA, TrkB, and p75NTR (D-H) did not differ across treatment conditions. Open circles represent males, closed circles represent females. Blot representations are in I. * indicates p < 0.05; # indicates a trending effect, p<0.06. Final N: control males (n = 8), control females (n = 7–8), AIE males (n = 7), AIE females (n = 8–9).

2.3. Acute Withdrawal: 24-Hour Time point

A. Prefrontal Cortex

As shown in Figure 4, 24-hrs following the last gavage, proNGF levels in the PFC were found to be significantly increased in AIE-treated rats compared to CON rats (F(1,28) = 5.855, p < 0.023; ηp2= 0. 0.184), but there was no sex difference (both p> 0.17). There were no differences in mature NGF levels as a function of Exposure (p = 0.18), Sex (p = 0.92), or the interaction with Sex (p = 0.53). In contrast, proBDNF expression was significantly lower in AIE rats compared to CON rats (F(1,28) = 4.436, p = 0.044; ηp2= 0.129); regardless of sex (both p’s >0.48). There were no differences in the expression of mBDNF, vAChT, TrkA; TrkB, or p75NTR as a function or interaction of Exposure and Sex (all p’s>0.09).

Figure 4: Prefrontal Cortex – 24 hours post AIE.

Figure 4:

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Protein expression of pro- and mature neurotrophins, neurotrophin receptors, and vAChT in the mPFC 24-hours following the last ethanol gavage, measured with western blot and ®-actin housekeeper. Graphs represent the group mean relative expression of protein compared to control ±SEM. proNGF protein expression was significantly higher in AIE treated animals 24 hours following the last gavage (A), but NGF expression did not differ across treatment conditions (B). proBDNF expression following AIE was significantly lower compared to CON rats (C), but BDNF protein expression did differ between CON and AIE (D). vAChT, TrkA, TrkB, and p75NTR protein expression did not significantly differ across treatment conditions in the PFC 24 hours following AIE (E-H). Open circles represent males, closed circles represent females. Blot representations are in I.

* Indicates p < 0.05. Final N: CON l males (n = 8), CON females (n = 7–9), AIE males (n = 8), AIE females (n = 7–9). Outlier removed: proNGF and NGF (n = 2 CON females, 2 AIE females), p75 (n = 1 CON female).

B. Nucleus Basalis Magnocellularis

Twenty-four hours following the last ethanol gavage (see Figure 5), no group differences were present with regards to the expression of proNGF in the NbM (all p’s > 0.36). There were also no Sex effect on NGF levels (both p’s>0.51); however, AIE treated rats trended towards less expression of NGF compared to controls (F(1,28) = 4.174, p = 0.051; ηp2= 0.13; Figure 5B). BDNF expression did not differ as a function of Exposure (p = 0.98), Sex (p = 0.33), or the interaction (p = 0.59). AIE-treated rats, regardless of sex (both p’s>0.24), had significantly greater proBDNF expression in the NbM relative to CON rats (F(1,28) = 8.873, p = 0.006; ηp2= 0.228). While group differences in neurotrophins were evident, no significant differences were detected in the expression of their receptors (all p>0.45), nor were there differences in ChAT expression (all p’s > 0.13).

Figure 5: Nucleus Basalis Magnocellularis – 24-hours post AIE.

Figure 5:

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Pro- and mature neurotrophin, neurotrophin receptors, and ChAT expression 24-hours following AIE measured with western blot with ®-actin housekeeper. Graphs depict mean relative protein expression compared to CON ±SEM. proNGF protein expression did not significantly differ across treatment groups (A), while there was a trending reduction in the protein expression of NGF in AIE treated animals (B). proBDNF protein expression was significantly higher in AIE treated animals (C), although BDNF expression was similar across CON and AIE treatment groups (D). ChAT, TrkA, TrkB, and p75NTR protein expression did not differ as a function of treatment condition (E-H). Open circles represent males, closed circles represent females. Blot representations are in I. *** indicates p < 0.005, # indicates a trending effect.

Final N: CON males (n = 7–8), CON females (n = 7–9), AIE males (n = 8), AIE females (n = 7–9). Outlier removed: proNGF and NGF (n = 2 CON females, 2 AIE females), proBDNF (n = 1 CON male, 2 AIE females), TrkA (n = 1 CON female, 1 AIE female), TrkB and p75 (n=1 AIE male).

2.4. Protracted Abstinence: 3-Week Time point

A. Prefrontal Cortex

Three weeks following the last ethanol gavage (see Figure 6), no Exposure or Sex differences in proNGF or mature NGF expression were found in the PFC (all p’s>0.82). There was a nonsignificant trend for a Sex effect on proBDNF expression (F(1,33) = 3.614, p = 0.066), where proBDNF expression in the PFC was slightly higher in males than in females. However, proBDNF did not differ across Exposure condition (both p’s>0.45). Expression of BDNF, TrkA, TrkB, p75NTR, or vAChT did not significantly differ across Exposure and/or Sex (all p’s>0.10). Thus, three weeks past the last ethanol dose, the effects of AIE on the PFC restabilized.

Figure 6: Medial Prefrontal Cortex – 3-weeks post AIE.

Figure 6:

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Protein expression of pro- and mature neurotrophins, their receptors, and vAChT 3-weeks following the last gavage, measured with western blot and with ®-actin housekeeper. Graphs depict mean relative protein expression compared to CON ±SEM. proNGF and NGF protein expression was similar across AIE and CON conditions (A-B). Similarly, proBDNF and BDNF expression did not change across treatment groups (C-D). vAChT, TrkA, TrkB, and p75NTR expression was not significantly different between CON and AIE treated rats (E-H). Blot representations are in I. Open circles represent males; closed circles represent females. Final N: CON males (n = 8), CON females (n = 9–10), AIE males (n = 8–9), AIE females (n = 9–10). Outlier removed: proNGF, BDNF and TrkB (n = 1 AIE female), NGF (n = 1 CON female), vAChT (n=1 AIE male)

B. Nucleus Basalis Magnocellularis

In prolonged abstinence, expression of mature NGF was significantly lower (F(1,32) = 6.777, p = 0.014; ηp2= 0.228) in AIE-treated rats compared to CON rats, regardless of Sex (p = 0.247; Figure 7 A, B). In contrast, NbM expression of proNGF did not differ across Exposure and Sex conditions (all p’s > 0.13). Furthermore, neither proBDNF nor mature BDNF differed significantly across conditions (all p’s>0.09). In addition, the expression of neurotrophin receptors, TrkA, TrkB and p75NTR did not differ across Exposure, Sex, or the interaction of the two factors (all p’s>0.10). Lastly, males and females did not differ in ChAT expression (p = 0.885). There was a trending reduction in the expression of ChAT in AIE treated rats compared to CON rats (F(1,33) = 4.002, p = 0.054; ηp2= 0.103; Figure 7 E–H).

Figure 7: Nucleus Basalis Magnocellularis – 3-weeks post AIE.

Figure 7:

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Neurotrophin and proneurotrophin protein expression, measured alongside neurotrophin receptors and ChAT 3- weeks following the last gavage. Data represent mean relative protein expression compared to CON measured though western blot with ®-actin housekeeper, ±SEM. While proNGF protein expression did not differ between treatment conditions in the NbM 3-weeks post AIE (A), NGF protein expression was significantly reduced in AIE treated animals (B). Neither proBDNF nor BDNF expression differed across treatment conditions (C-D). ChAT expression in the NbM was significantly lower in AIE treated animals compared to controls (E). TrkA, TrkB, and p75NTR proteins expression did not differ significantly across treatment groups (F-H). Blot representations are in I. Open circles represent males; closed circles represent females * indicates p < 0.05, # indicates a trending effect. Final N: CON males (n = 8), CON females (n = 8–9), AIE males (n = 9), AIE females (n = 8–9). Outlier removed: ChAT (n = 1AIE female), TrkA (n = 1 CON female).

2.5. Pearson’s Correlations

A. 2-hr Time point

Pearson’s correlations were run to explore the relationship between protein expression within and across brain regions in CON (Supplemental Figure 1A) and AIE-treated (Supplemental Figure 1B) rats 2-hrs following the last gavage. A summary of significant changes and correlations between neurotrophins and cholinergic markers are in Table 2. Within CON treated rats, it was found that PFC BDNF was positively correlated with PFC proBDNF (r(16) = 0.91, p < 0.0001) and vAChT (r(16) = 0.89, p <0.0001). In addition, proBDNF also positively correlated with PFC vAChT (r(16) = 0.86, p < 0.0001). Prefrontal cortical proBDNF positively correlated with PFC TrkB (r(16) = 0.81, p = 0.0002), and PFC TrkA positively correlated with PFC p75NTR (r(16) = 0.74, p<0.002). It was also found that NbM TrkA positively corelated with PFC TrkB (r(16) = 0.73, p = 0.0015). Lastly, TrkB positively correlated with p75NTR (r(16) = 0.77, p = 0.0005). In AIE treated animals PFC TrkA positively correlated with PFC proNGF (r(16) = 0.71, p = 0.002). Furthermore, in AIE treated animals, PFC BDNF expression is positively correlated with PFC vAChT (r(16) = 0.77, p = 0.0004).

Table 2.

Summary of critical findings for changes (↑ increase; ↓ decrease) and correlations as a function of treatment conditions (AIE or CON) and region (NbM or PFC).

NbM PFC Correlations with Cholinergic Markers
Intoxication (2-hrs post gavage) AIE ↓NGF AIE ↓BDNF
AIE ↓vAChT		 CON: PFC: BDNF/VAChT r= 0.89 (+)
CON: PFC: ProBDNF/VAChT r= 0.86 (+)
AIE: PFC: BDNF/VAChT r= 0.77(+)
AIE: PFC: ProNGF/TrkA r= 0.71 (+)
Withdrawal (24-hrs post gavage) AIE ↓NGF
AIE ↑ProBDNF		 AIE ↑ProNGF
AIE ↓ProBDNF		 CON: NbM: ProBDNF/ChAT r=0.79(+)
AIE: NbM: ProNGF/p75NTR r= 0.74(+)
Abstinence (3 wks post gavage) AIE ↓NGF
AIE ↓ChAT		 stable CON: PFC TrkA/NbM ChAT r=0.69 (−)
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B. 24-Hour Time point

Correlations were run to explore relationships between protein expression in CON (Supplemental Figure 2A) and AIE (Supplemental Figure 2B) rats within and between the PFC and NbM during the withdrawal period. In CON treated rats, PFC TrkB positively correlated with PFC BDNF (r(16) = 0.79, p = 0.0008). In addition, NbM ChAT positively correlated with NbM proBDNF expression (r(16) = 0.76, p = 0.0017). In AIE treated animals PFC proBDNF positively correlated with PFC BDNF expression (r(16) = 0.75, p = 0.0005) and PFC TrkB expression (r(16) = 0.74, p = 0.0006). In addition, PFC BDNF positively correlated with PFC TrkB expression (r(16) = 0.87, p < 0.0001). Lastly, NbM proNGF expression is positively correlated with NbM p75NTR (r(16) = 0.74, p = 0.0017).

C. 3-Week Time point

Exploratory correlations were run in CON (Supplemental Figure 3A) and AIE (Supplemental Figure 3B) rats, 3 weeks following the last gavage. A significant negative correlation (r(16) = −0.69, p = 0.0016) was found between NbM ChAT and PFC TrkA expression in control rats. However, no significant correlations were found in AIE treated animals at this time point.

Discussion

This study examined whether repeated binge ethanol exposure during adolescence disrupts the balance between pro and mature neurotrophins in a critical basal forebrain region and the frontal cortex, potentially contributing to the loss of basal forebrain cholinergic phenotype typically observed following this model. By examining changes in frontocortical and NbM neurotrophic profiles as a function of time from the last ethanol gavage, it is evident that there are unique and dynamic changes in both neurotrophin and proneurotrophin expression during intoxication, withdrawal, and protracted abstinence. More importantly, increases or decreases in the expression of proNGF and proBDNF did not necessarily reflect similar changes in the expression of their mature counterparts. In contrast, the expression of pro- and mature neurotrophin receptors did not change across intoxication, withdrawal, and abstinence. Interestingly, we found that during intoxication, expression of mature BDNF in the PFC, in both control rats and AIE rats, correlated with levels of VAChT in the PFC, a marker of cholinergic tone.

3.1. Neurotrophin expression is suppressed during ethanol intoxication:

Two hours following the last ethanol exposure, while BECs remain elevated, we observed decreases in BDNF, and vAChT in the prefrontal cortex, alongside reductions in NGF in the NbM. Since neurotrophins are synthesized and secreted in an activity-dependent manner by cortical pyramidal neurons and to a lesser extent by astrocytes and microglia, especially during an inflammatory response, (Bambrick et al., 2003; Becker et al., 2018; Elkabes et al., 1996; Marty et al., 1996; Marty et al., 1997; Minnone et al., 2017; Thoenen et al., 1991; Toyomoto et al., 2005) and are reduced by GABAa agonists, reductions in NGF and BDNF within the PFC were expected during ethanol intoxication. While our observed reductions in BDNF in the PFC replicate previously found reductions in BDNF expression during intoxication (Fernandez et al., 2017), NGF expression in the PFC was not reduced during intoxication. Transcriptional regulation of BDNF and NGF gene expression in response to alcohol may lead to differential BDNF and NGF expression. Although the driving force for the reduction in detected BDNF during intoxication was not pursued in this study, it could be due to reductions in gene transcription, reductions in protein maturation from pre-proBDNF to proBDNF to BDNF, or rather, an increase matrix metalloproteinase-9 (MMP-9) which degrades the mature NGF and BDNF. Based on previous work which compared the serum levels of MMP-9 in healthy adolescents compared to those admitted to the hospital for acute alcohol intoxication, it was found that acute intoxication significantly increased serum MMP-9 (Zdanowicz et al., 2022). Further evidence supports the notion that ethanol increases in MMP-9 expression were found following both acute and chronic intermittent ethanol exposure. Male Sprague Dawley rats exposed to 4 g/kg of 25% ethanol from PND 49 – 77 following a 2 day on 2 day off cycle, were found to have greater expression of MMP-9 in both the medial prefrontal cortex and the hippocampus (Yin et al., 2019). While further work should be done to include gene transcription of the neurotrophin family of proteins and their corresponding receptors during intoxication, the lack of changes in proBDNF and proNGF levels suggest that gene transcription may not be the driving force for reductions in mature BDNF and NGF expression. In addition, we did not see an accumulation of proBDNF or proNGF which would hint at neurotrophin maturational dysfunction. It appears that the reduction in neurotrophin expression at this stage of ethanol exposure may be due to increases in mature neurotrophin degradation mechanisms potentially driven by an increase in activity of extracellular matrix proteases.

However, another mechanism of ethanol’s action on neurotrophins is modulated through changes in microRNAs. There is evidence that chronic ethanol leads to changes in several microRNAs that control BDNF expression. In male ethanol-dependent adult rats, with BEC above 200, miR-206 was upregulated selectively in the PFC and led to escalated ethanol consumption (Tapocik et al, 2014). miR-206 is of interest because it represses BDNF expression. Furthermore, miR-30a-5p, which also regulates BDNF, when inhibited suppressed drinking and its suppression increased BDNF levels in adult male ethanol exposed mice (Darcq et al, 2015). These data demonstrate that ethanol-induced changes in microRNAs is a mechanism by which BDNF expression, and potentially NGF expression, is suppressed in the PFC by chronic high levels of ethanol.

3.2. Acute withdrawal alters pro-neurotrophin expression in a region-specific manner:

During acute withdrawal, 24 hours following the last ethanol gavage, dysregulation of immature and mature neurotrophins persist—but in a different pattern. In the PFC, proNGF levels are elevated in AIE-treated animals, while proBDNF expression is reduced. Conversely, in the NbM, 24-hrs post AIE, NGF expression was reduced, while proBDNF levels increased. The increase in proNGF and proBDNF expression across the PFC and NbM when compared to CON rats, agrees with previously published work examining chronic ethanol exposure (Popova et al., 2020). However, this region-specific change in neurotrophin expression is interesting considering that proBDNF expression is reduced in the PFC while simultaneously being elevated in the NbM. As the NbM does not locally produce neurotrophins, this elevated proBDNF most originates from several cholinergic projection regions such as the PFC, amygdala, anterior cingulate cortex (ACC), and OFC. The observed proBDNF elevation most likely reflects retrogradely transported proneurotrophins via dyneins along the cholinergic axons from the frontal cortex, or from the amygdala (Wu et al., 2014). It appears that AIE-induced reductions in proBDNF expression in the PFC parallels an increased proBDNF expression in the NbM. In contrast, AIE-induced elevations in proNGF in the PFC may contribute to reduced NGF in the NbM. The continued reduction of NGF within the NbM alongside elevations in proBDNF may propagate cholinergic degeneration, as the lack of mature trophic support makes cholinergic neurons susceptible to p75NTR mediated JNK/ c-JUN and RhoA downstream signaling (Bhakar et al., 2003) and degeneration in alcohol withdrawal.

3.3. Prefrontal cortical neurotrophin expression stabilizes, while NbM cholinergic markers are reduced in long-term abstinence:

Lastly, during prolonged abstinence (3 weeks post AIE) trophic balance in the prefrontal cortex appears to have been restored to homeostatic levels without long-lasting changes in the expression of neurotrophins, proneurotrophins, or their receptors. However, evidence of cholinergic degeneration in the NbM is present as ChAT expression is reduced in AIE animals compared to controls alongside reductions in NGF expression. The reduction in NGF expression in the NbM likely confers a state of greater vulnerability to further degeneration, as decreased detection of NGF in the NbM is suggestive of impairments in cholinergic NGF sequestration, which may result from a lack of access to target derived neurotrophins or deficits in axonal retrograde transport. Furthermore, the loss of ChAT expression is consistent with previous findings following AIE treatment where cholinergic phenotype markers are downregulated as cholinergic neurons enter a state of quiescence that may precede cell death if prolonged (Fernandez & Savage, 2017; Kipp & Savage, 2024; Vetreno et al., 2020; Vetreno et al., 2014; Vetreno & Crews, 2018).

The combination of findings at this time point poses an interesting question: If the neurotrophic profile is restored to homeostatic levels in the prefrontal cortex in AIE-treated animals, then why are there persistent reductions in NGF within the NbM; and why does the loss of cholinergic phenotype persist? This could potentially be the resultant impairments in NGF/BDNF retrograde transport. In a Ts65Dn model of Down’s syndrome, Cooper et al., (2001) observed dramatic impairments in retrograde transport of 125 I-NGF and atrophy of hippocampal projecting basal forebrain cholinergic neurons. Similarly, an in vitro model of senescent cholinergic neurons identified reductions in transport efficiency of pro- and mature neurotrophins (Fahnestock et al., 2021). Another possibility arises when considering that denervation of cholinergic terminals in neurotrophin rich cortical areas could be responsible for this pattern in findings. Denervation of cholinergic terminals would impede access of target derived neurotrophins for cholinergic neurons, leading to atrophy and reductions in cholinergic phenotype expression. The loss of target derived neurotrophins in this fashion would ultimately facilitate loss of cholinergic phenotype in a manner reminiscent of fimbria-fornix transection experiments (Gage et al., 1988; Hefti, 1986; Lazo et al., 2010). Recently, using a traumatic injury model, the fluid percussion injury (FPI) model, Dasgupta et al. (2023), demonstrated that cortical injury led to the induction of ipsilateral cortical proneurotrophin expression and subsequent degeneration of afferent basal forebrain cholinergic neurons. Moreover, cortical FPI in a p75NTR KO mouse line was unable to produce degeneration of afferent cholinergic neurons despite induction of cortical proneurotrophin expression. Finally, by culturing basal forebrain cholinergic neurons in a microfluidic chamber, it was demonstrated that application of proBDNF and proNGF in axonal compartments induced axon fragmentation (Dasgupta et al., 2023). Taken together, these findings suggest that AIE could induce cholinergic degeneration through a p75NTR dependent manner whereby basal forebrain cholinergic neurons retract from neurotrophin enriched projection sites. While this study provides circumstantial evidence of a proneurotrophic mechanism for reported cholinergic dysfunction during and after AIE, recent work from our lab has further examined this mechanism by pharmacologically manipulating p75NTR signaling during AIE. By inhibiting proneurotrophin related effects at p75NTR, using LM11A-31 during ethanol gavage, cholinergic neurons within the basal forebrain and associated attentional deficits were protected from loss due to AIE (Kipp & Savage, 2024).

3.4. Correlations supporting the role of neurotrophins in cell survival and pathology:

The positive correlation between mBDNF levels and vAChT in the PFC in both control and AIE rats supports the role of BDNF in maintaining cholinergic function (Orciani et al, 2022). Furthermore, the correlation is likely driven by the fact that during intoxication, AIE suppresses both BDNF and vAChT levels. During withdrawal, in AIE rats, proNGF expression positively correlated with NbM p75NTR, supporting a potential role for the interaction between the ligand and receptor in pathology (Fahnestock & Shekari, 2019). Other correlations observed during intoxication and withdrawal were mostly between ligands and their receptors or trophic receptors with each other within the PFC or NbM. During abstinence the only significant correlation was a negative one between NBM ChAT levels and TrkA receptors in the PFC in CON rats. This relationship is difficult to explain, but TrkA receptors are dynamic and are located on neurons, immune cells, as well as glia.

3.5. Future Directions and Limitations:

Future studies should examine dynein expression or rate of dynein transport in animal models using radiolabeled NGF (125I-NGF); however, cortical denervation itself could contribute to impairments in axonal transport. While we did not observe cortical reductions in vAChT at the 3-week timepoint in the PFC, we have found that vAChT is reduced in OFC 6 months post AIE, as well blunting of behaviorally elicited ACh release measured through in vivo microdialysis during a spontaneous alternation task in both the OFC and PFC (Fernandez & Savage, 2017; Kipp et al., 2021). Reductions of vAChT in the OFC may occur earlier than in the PFC, but further time points may be required to characterize the sequence of cholinergic marker loss across cortical regions. In addition, there should be an attempt to include the ACC as a potential site of pathology. While most NbM neurons terminate in only one cortical area, upwards of 20% of NbM cholinergic neurons have broader terminal fields and co-innervate the PFC, OFC, and ACC (Ballinger et al., 2016; Chandler et al., 2013). Additionally, we are unable to delineate the changes in neurotrophins, receptors, or cholinergic marker expression across prefrontal cortical subregions, such as prelimbic cortex, infralimbic cortex, and ACC, as all were part of the PFC tissue collected. Analysis of specific PFC subregions may shed light on unique ethanol-neurotrophin sensitivities within the PFC.

Future work should investigate the role of extracellular matrix proteins such as MMP’s in upregulating mature neurotrophin degradation, as well as the role of the tissue plasminogen activator (tPA)/plasmin impairments in conversion of proneurotrophins to mature neurotrophins, as changes of the NGF and BDNF maturational and degradative pathways likely contribute to the observed dysregulation of pro and mature neurotrophins during alcohol exposure. In a similar vein, markers for Trk receptor activation and downstream effectors of Trk and p75NTR activation such as AKT, ERK, or JNK would provide a valuable expansion on understanding neurotrophin dysregulation associated with adolescent alcohol use. In addition, changes in microRNAs that modulate neurotrophins should also be considered as several studies have shown that chronic ethanol exposure reduces microRNA that modulate BDNF levels, in at least adult animals (Darcq et al, 2015; Tapocik et al, 2014)

Males and females did not differ in expression patterns in most of our proteins of interest. However, it should be emphasized that males and females differed in NGF expression within the NbM, specifically 2-hours following the last gavage. Regardless of treatment condition, females had slightly greater NGF expression in the NbM compared to males. These findings did not extend through the other time points of interest, which may be due to sex differences in the expression of NGF regulatory enzymes in different brain regions that may be altered following ethanol exposure, which should be explored. In contrast, NbM proBDNF expression 2 hours post ethanol was higher in AIE-treated males than females. Although female rats metabolize alcohol more quickly (Marshland et al, 2021), and have higher levels of alcohol dehydrogenase activity (Quintanilla et al., 2007), no other differences between male and female AIE rats were identified during the intoxication period. Males with an AUD display increased levels of proBDNF, and levels of proBDNF may be a biomarker reflecting recent problematic alcohol use, at least in males (Zhou et al, 2010).

While these present findings shed light on the dynamic changes in proneurotrophin and mature neurotrophin expression in a region-specific manner during ethanol intoxication, withdrawal, and abstinence, it is necessary to take into consideration the limitations in the scope of our investigation. While the NbM projection to the PFC is a particularly relevant circuit to AUD pathology, western blot cannot effectively differentiate cholinergic from non-cholinergic neurons in these regions. Moreover, while a large portion of cholinergic neurons in the NbM innervate the frontal cortex, we could not isolate the subset of cholinergic neurons projecting to the PFC. One strength of western blotting presented here is the effective differentiation of pro and mature neurotrophins. While protein detection assays such as ELISA or IHC rely solely on antibody specificity for the detection of protein presence, the separation of proteins by weight in western blotting provides a reliable control against concerns over antibody reliability. As evident in this study, the same antibody was able to detect both the immature and mature forms of NGF and BDNF, and ELISA has been shown to have difficulty differentiating between pro and mature NGF (Fahnestock et al., 2001).

It should also be noted that we did identify that AIE treatment led to significant reductions in body weight over the course of treatment compared to controls. While both CON and AIE treated males and females significantly increased body weights throughout adolescence, AIE treatment reduced the average body weight in comparison to controls. On average, AIE treated males and females were roughly 9% and 4% smaller than their CON counterparts respectively. Whether this loss of body weight during AIE is a factor in pathological responses is unknown. However, work from our lab and others have consistently identified loss of cholinergic markers in the basal forebrain and frontal cortex without significant differences in body weight following AIE (Fernandez & Savage, 2017; Galaj et al., 2019; Reitz et al., 2021; Vetreno et al., 2020; Vetreno & Crews, 2012; Vetreno & Crews, 2018).

Taken together these findings highlight the potential role of neurotrophin dysregulation in mediating pathology because of adolescent alcohol intoxication and withdrawal. Similar to the kindling hypothesis of alcohol-related brain damage, it appears that adolescent binge ethanol exposure may induce cholinergic degeneration through cycles of alcohol intoxication and withdrawal (Spear, 2018). Where intoxication reduces mature neurotrophin signaling and withdrawal-induced increases in pro neurotrophin signaling. This pro/mature neurotrophin adaptation pattern may contribute to the loss of cholinergic phenotype expression in the basal forebrain, reductions in cortical ACh release, and cognitive impairment.

Supplementary Material

Supplement Fig 1

Supplemental Figure 1: Correlations in protein expression – 2-hr post AIE. Pearson’s correlation in protein expression within and between the NbM and PFC in CON and AIE treated rats. In CON treated rats, PFC vAChT positively correlated with proBDNF and BDNF expression in the PFC. In addition, proBDNF positively correlated with BDNF, whereas PFC p75NTR positively correlated with p75NTR. NbM TrkA positively correlated with PFC TrkB and NbM p75NTR positively correlated with NbM TrkB (A). In AIE treated rats, PFC TrkA positively correlated with PFC proNGF expression. PFC BDNF is also positively correlated with PFC vAChT expression (B).

Supplement Fig 2

Supplemental Figure 2: Correlations in protein expression – 24hr post AIE. Pearson’s correlation in protein expression within and between the NbM and PFC in CON and AIE treated rats. Twenty-four hours following the last gavage, PFC TrkB positively correlated with PFC BDNF, and NbM ChAT positively correlated with NbM proBDNF (A). In AIE treated animals, PFC TrkB expression positively correlated with PFC proBDNF and BDNF expression. Moreover, NbM proNGF positively correlated with PFC p75NTR expression (B).

Supplement Fig 3

Supplemental Figure 3: Correlations in protein expression – 3 weeks post AIE. Pearson’s correlation in protein expression within and between the NbM and PFC in CON and AIE treated rats. Three weeks following the last ethanol gavage, in protracted abstinence, NbM ChAT negatively correlated with PFC TrkA (A). After controlling for multiple contrasts, no significant correlations were found between protein expression in the NbM and PFC of AIE treated rats (B).

Supplement Fig 4

Supplemental Figure 4: PFC 2-hr Time point Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, vAChT, and TrkB (B)

Supplement Fig 5

Supplemental Figure 5: NbM 2-hr Time point Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, ChAT, and TrkB (B).

Supplement Fig 6

Supplemental Figure 6: PFC 24-hr Time point Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, vAChT, and TrkB (B).

Supplement Fig 7

Supplemental Figure 7: NbM 24-hr Time point Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, ChAT, and TrkB (B).

Supplement Fig 8

Supplemental Figure 8: PFC 3-Week Timepoint Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, vAChT, and TrkB (B).

Supplement Fig 9

Supplemental Figure 9: NbM 3-Week Timepoint Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, ChAT, and TrkB (B).

Sources of Support:

The authors disclosed receipt of the following financial support for their research, authorship and/or publication of this article: This work was supported by UO1 AA028710 (LMS); P50 AA017823 (LMS); BTK was supported by T32 AA025606 (JDJ).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement Fig 1

Supplemental Figure 1: Correlations in protein expression – 2-hr post AIE. Pearson’s correlation in protein expression within and between the NbM and PFC in CON and AIE treated rats. In CON treated rats, PFC vAChT positively correlated with proBDNF and BDNF expression in the PFC. In addition, proBDNF positively correlated with BDNF, whereas PFC p75NTR positively correlated with p75NTR. NbM TrkA positively correlated with PFC TrkB and NbM p75NTR positively correlated with NbM TrkB (A). In AIE treated rats, PFC TrkA positively correlated with PFC proNGF expression. PFC BDNF is also positively correlated with PFC vAChT expression (B).

NIHMS2001864-supplement-Supplement_Fig_1.pdf (514.8KB, pdf)
Supplement Fig 2

Supplemental Figure 2: Correlations in protein expression – 24hr post AIE. Pearson’s correlation in protein expression within and between the NbM and PFC in CON and AIE treated rats. Twenty-four hours following the last gavage, PFC TrkB positively correlated with PFC BDNF, and NbM ChAT positively correlated with NbM proBDNF (A). In AIE treated animals, PFC TrkB expression positively correlated with PFC proBDNF and BDNF expression. Moreover, NbM proNGF positively correlated with PFC p75NTR expression (B).

NIHMS2001864-supplement-Supplement_Fig_2.pdf (513.5KB, pdf)
Supplement Fig 3

Supplemental Figure 3: Correlations in protein expression – 3 weeks post AIE. Pearson’s correlation in protein expression within and between the NbM and PFC in CON and AIE treated rats. Three weeks following the last ethanol gavage, in protracted abstinence, NbM ChAT negatively correlated with PFC TrkA (A). After controlling for multiple contrasts, no significant correlations were found between protein expression in the NbM and PFC of AIE treated rats (B).

NIHMS2001864-supplement-Supplement_Fig_3.pdf (544.2KB, pdf)
Supplement Fig 4

Supplemental Figure 4: PFC 2-hr Time point Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, vAChT, and TrkB (B)

NIHMS2001864-supplement-Supplement_Fig_4.pdf (408.1KB, pdf)
Supplement Fig 5

Supplemental Figure 5: NbM 2-hr Time point Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, ChAT, and TrkB (B).

NIHMS2001864-supplement-Supplement_Fig_5.pdf (426.5KB, pdf)
Supplement Fig 6

Supplemental Figure 6: PFC 24-hr Time point Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, vAChT, and TrkB (B).

NIHMS2001864-supplement-Supplement_Fig_6.pdf (447.2KB, pdf)
Supplement Fig 7

Supplemental Figure 7: NbM 24-hr Time point Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, ChAT, and TrkB (B).

NIHMS2001864-supplement-Supplement_Fig_7.pdf (354.3KB, pdf)
Supplement Fig 8

Supplemental Figure 8: PFC 3-Week Timepoint Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, vAChT, and TrkB (B).

NIHMS2001864-supplement-Supplement_Fig_8.pdf (544.3KB, pdf)
Supplement Fig 9

Supplemental Figure 9: NbM 3-Week Timepoint Western Blots. Raw western blot strips are presented alongside ladder. Blots were cut horizontally at the indicated ladder markings. Representative western blot strips for the analysis of NGF, proNGF, Beta-actin, p75NTR, and TrkA (A) and BDNF, proBDNF, Beta-actin, ChAT, and TrkB (B).

NIHMS2001864-supplement-Supplement_Fig_9.pdf (489.7KB, pdf)

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