Dysregulation of neurotrophin expression in prefrontal cortex and nucleus basalis magnocellularis during and after adolescent intermittent ethanol exposure

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 5 g/kg 20% EtOH or water gavage on two-day-on, two-day-off cycles from post-natal day 25-57. Rats were sacrificed 2 h, 24 h, 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: Abstinence; Adolescent; Ethanol; Neurotrophins; Withdrawal.

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

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 8^th 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)

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

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).

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

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).

Figure 4:. Prefrontal Cortex – 24 hours post AIE.

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).

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

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).

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

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)

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

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).