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. Author manuscript; available in PMC: 2022 Feb 1.
Published in final edited form as: Birth Defects Res. 2020 Nov 10;113(3):299–313. doi: 10.1002/bdr2.1839

Examination of cortically projecting cholinergic neurons following exercise and environmental intervention in a rodent model of fetal alcohol spectrum disorders

Katrina A Milbocker 1, Anna Y Klintsova 1
PMCID: PMC8274426  NIHMSID: NIHMS1718236  PMID: 33174398

Abstract

Background:

Up to 1 in 5 infants in the United States are exposed to alcohol prenatally, resulting in neurodevelopmental deficits categorized as fetal alcohol spectrum disorders (FASD). Choline supplementation ameliorates some deficits, suggesting that alcohol exposure (AE) perturbs cholinergic neurotransmission and development. Behavioral interventions, which upregulate cholinergic neurotransmission, rescue cognitive deficits in rodent models of FASD.

Methods:

We investigated the impacts of two interventions (either wheel-running (WR) or “super intervention,” WR plus exposure to a complex environment) on cholinergic neuronal morphology in the nucleus basalis of Meynert (NBM), the source of cortical cholinergic input, and prefrontal cortex (PFC) in a rodent model of FASD. One third of the total 47 male pups received intragastric intubation of ethanol in milk substitute during postnatal days (PD) 4–9. Another third served as sham-intubated procedural controls while the final third served as suckle controls. Rats from each group were exposed to either intervention during PD 30–72. Choline acetyltransferase (ChAT+) and acetylcholinesterase staining were used to quantify cholinergic neuron number, soma volume, and axon number.

Results:

Our data indicate a main effect of postnatal treatment on ChAT+ neuron number in NBM in adulthood. Post hoc analysis demonstrates that ChAT+ neuron number is reduced in AE compared to suckle control rodents (p < .01).

Conclusions:

We examined the cytoarchitectonics of cholinergic neurons in NBM and PFC in adulthood following early postnatal AE and two interventions. We show that AE reduces ChAT+ neuron number in NBM, and this is not mitigated by either intervention.

Keywords: choline, environmental complexity, exercise, fetal alcohol spectrum disorders, nucleus basalis of Meynert

1|. INTRODUCTION

Up to 1 in 5 infants in the United States has been exposed to alcohol prenatally, resulting in impaired development of the nervous system and ultimately impacting neurocognitive capabilities (May et al., 2015). The range of deficits stemming from alcohol teratogenicity is due to the timing and amount of alcohol exposure (AE), leading to a variety of disorders categorized under the umbrella term fetal alcohol spectrum disorders (FASD) (Livy, Maier, & West, 2001; May et al., 2013). AE during the brain growth spurt, a period of rapid brain growth and synaptogenesis occurring during the third trimester of human pregnancy and first two postnatal weeks in rodents (Dobbing & Sands, 1979), can produce lasting impairments in cortex-dependent behaviors. Specifically, AE during the brain growth spurt results in executive function deficits which are primarily driven by activation of the prefrontal cortex (PFC). These executive function behaviors include decision-making and inhibitory control (Connor, Sampson, Bookstein, Barr, & Streissguth, 2000; Driscoll, Streissguth, & Riley, 1990; Kable et al., 2020; Sowell et al., 2001).

The mammalian brain cholinergic system is the largest continuous aggregate of neurons in the central nervous system, consisting of six to eight major nuclei. Neurotransmission of acetylcholine is important for many cognitive capacities including learning, memory, consciousness, and sleep. The nucleus basalis of Meynert (NBM) is the primary source of cholinergic input to the mammalian cortical mantle, including the PFC (Karczmar, 2007). The developing cholinergic system is vulnerable to environmental neurotoxicants and nutrient deficiency (Derbyshire & Obeid, 2020; Eriksson, Ankarberg, Viberg, & Fredriksson, 2001; McKeon-O’Malley, Siwek, Lamoureux, Williams, & Kowall, 2003; Wallace, Blusztajn, Caudill, Klatt, & Zeisel, 2019). It has been demonstrated that prenatal and early postnatal AE perturbs natural choline metabolism and cholinergic signaling (acetylcholine) in the forebrain, resulting in an altered epigenetic profile, enzymatic dysfunction (of synthesizing choline acetyltransferase, ChAT, and hydrolyzing enzyme acetylcholinesterase, AChE), and likely causing cellular apoptosis due to a lack of available choline (Fagerlund et al., 2006; Light, Serbus, & Santiago, 1989; O’Neill et al., 2019; Zeisel, 2011). Further, AE during the brain growth spurt in rodents alters the density and binding affinity of certain cholinergic receptors in the adult rodent hippocampus (Kelly, Black, & West, 1989; Monk, Leslie, & Thomas, 2012) and impairs memory.

Insufficient maternal choline intake during the perinatal period has been shown to exacerbate the negative impact of prenatal and early postnatal AE on offspring behavior and cognition in a rodent model of FASD (Idrus, Breit, & Thomas, 2017). Further, antagonism of cholinergic neurotransmission through systemic (physostigmine, an acetylcholinesterase inhibitor) and intracerebral (scopolamine, a muscarinic cholinergic receptor antagonist) pharmacological manipulation produces similar behavioral deficits in rodents, implicating cholinergic signaling dysfunction in FASD pathology (Heroux, Horgan, Rosen, & Stanton, 2019; Robinson-Drummer, Heroux, & Stanton, 2017). Thus, there exists substantial evidence proving that AE during the brain growth spurt leads to dysfunction of cholinergic neurons, ultimately contributing to cognitive deficits.

Dietary choline supplementation, an essential nutrient with a wide range of functions in metabolism (from cellular structure to neurotransmitter synthesis), has been an attractive target for the development of a non-invasive intervention strategy for children diagnosed with FASD. Preclinical rodent studies indicate that choline supplementation concurrent with early postnatal AE or in adolescence reverses damage to the global epigenome of the hippocampus and PFC in addition to ameliorating cognitive and behavioral deficits in AE rats (Balaraman, Idrus, & Thomas, 2017; Bottom, Abbott, & Huffman, 2020; Davis, Tang, He, Lee, & Bearer, 2020; Otero, Thomas, Saski, Xia, & Kelly, 2012; Perkins, Fadel, & Kelly, 2015; Ryan, Williams, & Thomas, 2008; Schneider & Thomas, 2016). Clinical studies assessing the efficacy of maternal choline supplementation during the perinatal period demonstrate a transient increase in infant cognition speed and an increase in visual recognition memory in FASD-diagnosed infants (Caudill, Strupp, Muscalu, Nevins, & Canfield, 2018; Fuglestad et al., 2013, 2015; Jacobson et al., 2018; Wozniak et al., 2020). However, this outcome is inconsistent and is most efficacious when administered either during prenatal AE or prior to 5 years of age (Nguyen, Risbud, Mattson, Chambers, & Thomas, 2016).

In addition to choline supplementation, behavioral interventions are known to upregulate cholinergic neurotransmission and strengthen synaptic connections in salient rodent brain cholinergic pathways (Bennett, Krech, & Rosenzweig, 1964; Diamond, Krech, & Rosenzweig, 1964; Tees, 1999; Woolf, 1991). Aerobic activity via free access to running wheels stimulates cholinergic neurotransmission in the normative and diseased brain, leading to altered hippocampal theta-rhythm synchrony and increases the production of growth factors (brain derived neurotrophin factor and nerve growth factor) which are necessary for synaptic re-modeling (Berchtold, Patrick Kesslak, & Cotman, 2002; Cotman, Berchtold, & Christie, 2007; Hall, Gomez-Pinilla, & Savage, 2018; Hall & Savage, 2016; Knipper et al., 1994; Neeper, Gómez-Pinilla, Choi, & Cotman, 1996; Vanderwolf, 1968; Voss, Vivar, Kramer, & van Praag, 2013). Voluntary wheel-running (WR) in adulthood has been shown to rescue reductions to cholinergic neuron number and size in the basal forebrain following adolescent AE (Vetreno et al., 2020). In addition to aerobic exercise, exposure to a complex environment (environmental complexity, EC) improves memory acquisition and consolidation via increased cholinergic signaling (Galaj, Barrera, & Ranaldi, 2020; Murphy, Foley, O’Connell, & Regan, 2006; Paban, Chambon, Jaffard, & Alescio-Lautier,-2005). The synergistic effects of a “super-intervention,” exposure to voluntary WR prior to inhabitation in EC, has been shown to improve neuroanatomical, epigenetic, and motor impairments in rodent models of FASD (Boschen, McKeown, Roth, & Klintsova, 2017; Hamilton, Criss, & Klintsova, 2015; Hamilton, Whitcher, & Klintsova, 2010; Klintsova et al., 1998; Wagner, Klintsova, Greenough, & Goodlett, 2013; Whitcher & Klintsova, 2008). However, the interactive effects of either voluntary exercise or “super-intervention” and AE during the brain growth spurt on the cytoarchitectonics of cortically-projecting cholinergic neurons remains unknown.

Our study investigates the effects of two non-invasive behavioral interventions (WR and environmental complexity) on the morphology of cortical afferentation by cholinergic neurons in a rodent model of FASD. The major source of cortical cholinergic innervation originates in NBM in the mammalian basal forebrain. NBM-cortical projections are responsible for cortical–cortical and cortical–subcortical theta synchrony necessary for attention control, sensory perception and integration, and movement coordination (Bloem et al., 2014; Eckenstein, Baughman, & Quinn, 1988; Hosseini, Alaei, Reisi, & Radahmadi, 2017; Karczmar, 2007; Meck, Williams, Cermak, & Blusztajn, 2008; Mesulam & Geula, 1988; Sarter & Lustig, 2020). Given the impact of early postnatal AE and behavioral intervention on cholinergic neurotransmission, we hypothesized that these factors would have an impact on the morphology of cholinergic neurons and their axons in NBM and PFC in adulthood.

2|. MATERIALS AND METHODS

2.1|. Experimental subjects

All animal procedures were approved by the University of Delaware’s Institutional Animal Care and Use Committee and performed in accordance with NIH’s Animal Care Guidelines. Timed-pregnant Long-Evans dams were obtained from Harlan Laboratories (Indianapolis, IN). Rats were maintained on a 12-hr light/dark cycle with light onset at 09:00 in a temperature-controlled colony room. On postnatal day (PD) 3, litters were culled to eight pups each, consisting of six males and two females when possible. This experiment includes the examination of brain tissue from 47 male rats from litters generated for a larger study (Boschen et al., 2017). As such, group size consists of 4–6 animals per postnatal treatment group/intervention group (included in all graphs). The impact of sex as a risk factor for FASD-related neuropathology is controversial, however several rodent studies indicate that regions of the male brain are more vulnerable to alcohol-induced neuroanatomical alteration compared to the female brain (Coleman, Oguz, Lee, Styner, & Crews, 2012; Ruggiero, Boschen, Roth, & Klintsova, 2018; Terasaki, Blades, & Schwarz, 2015).

2.2|. Early postnatal AE and blood alcohol concentration analysis

On PD 4, pups were randomly assigned to one of three postnatal treatment groups: AE, sham-intubated (SI), or suckle control (SC). AE pups received alcohol in milk substitute (5.25 g kg−1 day−1 at 11.9% vol/vol) through intragastric intubation twice daily 2 hr apart on PD 4–9 during the rodent brain growth spurt (Dobbing & Sands, 1979). SI pups were intubated twice daily on PD 4–9, but did not receive any liquids during the treatment period to control for the stress of intubation. SC pups were left undisturbed with the dams. Pups from all postnatal treatment groups were weighed daily, and AE pups received a third milk-only dose 2 hr after the second alcohol intubation to prevent a decline in nutritional health due to binge AE on PD 4–9. Blood samples (60 μl) were acquired via tail clip on PD 4, 90 min following the second alcohol dose for analysis of peak blood alcohol concentration (BAC) in AE and procedural control (SI) pups (Bonthius & West, 1990). Plasma was discarded for SI pups and analyzed for BAC of AE pups using an Analox GL5 Alcohol Analyzer (Analox Instruments, Boston, MA). Due to a machine malfunction, plasma from one AE animal used in this study was not analyzed. Following postnatal treatment, pups were left undisturbed until weaning on PD 23 when animals were socially housed (SH) in same-sex groups of three, counterbalanced for litter and postnatal treatment.

2.3|. Non-Invasive behavioral interventions

2.3.1|. Voluntary WR

On PD 30, one third of animals from all postnatal treatment groups was transferred to modified cages with free access to a stainless steel running wheel in groups of three while a second third of rats remained in control SH cages (Helfer, Goodlett, Greenough, & Klintsova, 2009; Van Praag, Christie, Sejnowski, & Gage, 1999). Running distance was recorded daily at 09:00.

2.3.2|. “Super-intervention”: Wheel running followed by exposure to a complex environment

The final third of animals began the “super-intervention,” wheel running followed by exposure to a complex environment (WREC), on PD 30. WREC animals began in WR cages (described previously) where they had free access to running wheels until PD 42 when they were transferred to the highly stimulating environmentally complex (EC) cages, free access to running wheels followed by exposure to a complex environment, as described in Gursky and Klintsova (2017). Wheels were checked daily at 09:00 from PD 30–42 to measure the distance run. Animals from all intervention and control groups were sacrificed on PD 72 and brain tissue was collected (see experimental timeline in Figure 1).

FIGURE 1.

FIGURE 1

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Experimental timeline for animals from all three postnatal conditions (alcohol exposure [AE], sham-intubated [SI], suckle control [SC]) exposed to either socially housed (SH) control, wheel running (WR) intervention, or “super-intervention” (WREC) from postnatal days (PD) 30–72

2.4|. Immunocytochemical and histochemical staining

On PD 72, all animals were sacrificed and transcardially perfused with 100 ml 0.1 M Phosphate Buffer Solution (PBS, pH = 7.20 at 4°C) followed by 100 ml 4% paraformaldehyde in PBS (pH = 7.20 at 4°C). Brain tissue was extracted and post-fixed in a 30% sucrose-paraformaldehyde solution for cryoprotection and stored at 4°C. Brain tissue was sectioned horizontally at 40 μm and stored maintaining sections’ order in cryoprotectant solution at −20°C.

2.4.1|. Choline acetyltransferase (ChAT+) immunocytochemical stain

A primary monoclonal antibody against choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, was used for the visualization of cholinergic neurons in the basal forebrain NBM. Every eighth tissue section was collected throughout the region of interest yielding four to six (rostro-caudal) tissue sections per brain. Free-floating tissue sections were triple washed in 1X Tris buffered saline (TBS) solution for 5 min/wash. Next, sections were incubated in a solution containing 0.4% Triton X-100 and 0.6% hydrogen peroxide in TBS for half an hour followed by three 5-min TBS washes. Sections were then transferred to blocking solution (3% normal bovine serum and 0.4% Triton X-100 in TBS) for 1 hr. Finally, sections were incubated in primary anti-ChAT antibody solution (anti-rabbit AB144P [Millipore-Sigma], 1:200 dilution in blocking solution) overnight at 4°C.

The following day, sections were triple washed in TBS for 5 min each. Tissue was then incubated in the secondary antibody solution (biotinylated anti-goat BA-9500 [Millipore-Sigma], 1:200 dilution in blocking solution) for 2 hr followed by triple wash and incubation in ABC-blocking solution for 1 hr. Sections were then triple washed in TBS before incubation in 0.05% 3,3′-Diaminobenzidine (DAB)-Peroxidase solution for visualization of neurons using brightfield microscopy (Figures 2a and S1). Sections were mounted onto doublesubbed slides in TBS and cover-slipped with DPX Mounting media.

FIGURE 2.

FIGURE 2

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(a) Appearance of ChAT+ neurons in nucleus basalis of Meynert (NBM) at high magnification (×40). (b) Total number of ChAT+ neurons in NBM of animals from three postnatal treatments (suckle control [SC], sham-intubated [SI] and alcohol exposure [AE]) and three intervention groups (socially housed [SH], wheel-running [WR] and WREC) in adulthood [* represents p < .05]. (c) Volume of ChAT+ neurons in NBM of animals from three postnatal treatments (SC, SI and AE) and three intervention groups (SH, WR and WREC) in adulthood. (d) Structural volume of NBM in animals from three postnatal treatments (SC, SI and AE) and three intervention groups (SH, WR and WREC) did not differ significantly in adulthood. n = 4–6 pups/postnatal treatment group/intervention group; error bars represent SEM

2.4.2|. Acetylcholinesterase (AChE+) histochemical stain

A Hedreen and Bacon modified Karnovsky-Roots method (Hedreen, Bacon, & Price, 1985) to localize AChE-containing fibers was performed on every eighth tissue section for the visualization of cholinergic fibers in the prelimbic sub-region of the medial PFC (Cg2, Paxinos and Watson Atlas, 2014). Four to five tissue sections were analyzed rostral to caudal through the region of interest. First, free-floating sections were rinsed twice with two 0.1 M sodium acetate buffer for 5 min each. Sections were then incubated in 0.1 mM acetylthiocholine medium (19% dH2O, 4% 0.1 M sodium citrate, 10% 0.03 M cupric sulfate, 2% 0.005 M potassium ferricyanide, and 25 mg S-acetylthiocholine iodide [Acros Organics] in 0.1 M sodium acetate buffer) for 2 hr. Tissue was again rinsed in 0.1 M sodium acetate buffer and transferred into a 4% ammonium sulfide solution for 15 min. Next, sections were exposed to five 1-min 0.1 M sodium nitrate washes and incubated in 0.1% silver nitrate solution for 15 min. Finally, tissue was double-rinsed in 0.1 M sodium acetate buffer for 5 min each before sections were mounted onto doublesubbed slides and cover-slipped with DPX Mounting media (Figures 3a and S2).

FIGURE 3.

FIGURE 3

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(a) AChE+axons in Cg2 at high magnification (×100). (b) AChE+ fiber number in Cg2 of mPFC of animals from three postnatal treatments (suckle control [SC], sham-intubated [SI] and alcohol exposure [AE]) and three intervention groups (socially housed [SH], wheel-running [WR] and WREC) did not differ significantly in adulthood. (c) Structural volume of Cg2 (prelimbic medial prefrontal cortex [PFC]) in animals from three postnatal treatments (SC, SI and AE) and three intervention groups (SH, WR and WREC) did not differ significantly in adulthood. n = 4–6 pups/postnatal treatment group/intervention group; error bars represent SEM

2.5|. Morphological analysis via unbiased stereology

2.5.1|. Estimation of cholinergic neuronal morphology

Estimation of total neuron and axon number was quantified using a Zeiss Axioskop 2 plus microscope (Carl Zeiss Inc., Thornwood, NY) and the Optical Fractionator probe in StereoInvestigator software (MBF Bioscience, Williston, VT) (Gundersen, 1986; Sterio, 1984; West, Slomianka, & Gundersen, 1991; West, 2002). Neuronal volume estimation was quantified using the Nucleator probe in StereoInvestigator software (Gundersen, 1988). For neuron number estimation, NBM was first manually outlined on all selected sections using a 5X objective and using the corpus callosum, striatum, and hippocampus as anatomical landmarks from Bregma −6.38 to −7.60 in accordance with the Paxinos and Watson Atlas, (2014). A 40X objective was then used to quantify the number and measure the volume of ChAT+ neurons throughout the depth of NBM in every eighth mounted tissue section with a counting frame and grid size of 200 μm × 200 μm, disector height of 25 μm, and 2 μm guard zone to account for tissue shrinkage.

Fiber number was similarly estimated. The Cg2 sub-region of the medial PFC was outlined using the corpus callosum, striatum, and hippocampus as anatomical landmarks from Bregma −3.10 to −4.10 in accordance with the Paxinos and Watson Atlas, (2014). A 100X oil immersion objective was then used to estimate fiber number throughout the depth Cg2 in every eighth mounted tissue section with a counting frame of 10 μm × 10 μm, a grid size of 250 μm × 250 μm, a disector height of 20 μm and guard zone of 2 μm. All number and volume estimations were validated by comparing mean coefficients of error (CE) for each parameter per animals. Mean CE’s for all parameters were between 0.01 and 0.09, consistently below the threshold for stereological exclusion, 0.1 (Glaser & Wilson, 1998).

2.6|. Statistical analyses

2.6.1|. Animals weights and BAC

Animal weights for each day of dosing (PD 4–9) and pre and post-intervention (PD 30 and 72) were averaged across postnatal treatment condition/intervention group at each time point. PD 4–9 weights were analyzed using a repeated-measures analysis of variance (ANOVA) and PD 30 and 72 weights were analyzed with a two-way (postnatal treatment × intervention group) ANOVA. Average PD 4 BACs for each animal were calculated based on 2–3 analyses per sample and the means for the AE group are reported as mg/dl ± SEM. Litter effects were controlled as no more than one animal per litter was assigned to a particular postnatal treatment and intervention condition. Differences were considered to be statistically significant at p < .05 and tests were followed by Tukey’s post hoc test when appropriate. Statistical analyses were run using SPSS IBM (v26).

2.6.2|. Cholinergic neuronal morphology

Cholinergic neuronal soma number, soma volume, axonal number, and overall volume of NBM and Cg2 were analyzed using two-way (postnatal treatment x intervention group) ANOVA. Differences were considered to be statistically significant at p < .05 and tests were followed by Tukey’s post hoc test when appropriate. A summary of the main neuroanatomical findings is represented in Table 1.

TABLE 1.

Summary of the effects of developmental alcohol exposure and/or behavioral intervention (voluntary WR or “super-intervention”) on NBM-cortical cholinergic neurons as demonstrated by two-way ANOVA analyses

Measure Brain region Postnatal treatment Intervention Interaction Sig. post hoc
ChAT+ neuron number NBM * ns ns AE vs. SC
ChAT+ neuronal volume NBM ns ns ns ns
Structural volume NBM, Cg2 ns, ns ns, ns ns, ns ns, ns
AChE+ fiber number Cg2 ns ns ns ns
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Abbreviations: AE, alcohol exposure; ANOVA, analysis of variance; NBM, nucleus basalis of Meynert; SC, suckle control; WR, wheel-running.

*

p < .05.

3|. RESULTS

3.1|. Animal weights

A comparison of animal weights from PD 4–9 indicates that AE significantly reduces pup weight over the treatment period. For PD 4–9, a postnatal day × postnatal treatment interaction was found (F[10,44] = 12.364, p < .0001, ηp2 = 0.360), as well as main effects of postnatal day (F[5,44] = 1,112.62, p < .0001, ηp2 = 0.962) and postnatal treatment (F[2,44] = 8.67, p = .001, ηp2 = 0.283). One-way ANOVAs on PD 4 and 9 weights determined that there was no effect of postnatal treatment on PD 4 (p > .1), however, AE animals weighed less than SI and SC pups on PD 9 (main effect of postnatal treatment: F[2,46] = 12.04, p < .0001, ηp2 = 0.201; Tukey’s post hoc: p < .002 for AE vs. SC and AE vs. SI). On PD 9, SI and SC pup weight did not differ (p = .567).

An alcohol-induced reduction in weight was not sustained into adolescence and adulthood. On PD 30, there were no significant main effects of postnatal treatment (F[2,38] = 1.789, p = .181, ηp2 = 0.086) or intervention group (F[2,38] = 0.499, p = .611, ηp2 = 0.026) on animal weight status. The interaction between postnatal treatment × intervention group was not significant (p > .1). Similarly, on PD 72 there were no significant main effects of postnatal treatment (F[2,38] = 0.783, p = .465, ηp2 = 0.043) or intervention group (F[2,38] = 2.184, p = .128, ηp2 = 0.111) on animal weight status. The interaction between postnatal treatment × intervention group was not significant (p > .1). Therefore, AE during the brain growth spurt resulted in an immediate, yet transient, reduction in animal weight in our experiment and neither intervention caused a change in animal weight (Table 2).

Table 2.

Summary of mean weights on postnatal days (PD) 4, 9, 30, and 72 of animals from all postnatal treatment (SC, SI, AE) and intervention groups (SH, WR, WREC)

Postnatal treatment PD Mean weight (g ± SD)
SH WR WREC
AE 4 10.85 ± 0.72 n/a n/a
9 17.38 ± 1.82* n/a n/a
30 96.40 ± 5.32 99.00 ± 5.02 98.00 ± 10.32
72 379.40 ± 31.05 363.50 ± 15.40 380.60 ± 18.97
SI 4 10.55 ± 1.02 n/a n/a
9 19.49 ± 1.64* n/a n/a
30 101.20 ± 6.57 105.25 ± 4.57 100.33 ± 7.26
72 388.00 ± 9.25 375.50 ± 11.00 386.50 ± 16.98
SC 4 10.51 ± 0.96 n/a n/a
9 20.11 ± 1.49* n/a n/a
30 104.20 ± 6.65 99.50 ± 4.04 98.50 ± 6.89
72 399.00 ± 43.31 373.25 ± 10.66 379.75 ± 28.29
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Notes: Significant between group differences are represented with *.

Abbreviations: AE, alcohol exposure; SC, suckle control; SH, socially housed; SI, sham-intubated; WR, wheel-running.

3.2|. Blood alcohol concentration

BACs were analyzed in plasma collected on PD 4, 90 min following the second alcohol dose. The BACs averaged 369.88 mg/dl (±18.00 SEM). This value is in the range of acceptable BACs for our binge drinking model (Boschen, Hamilton, Delorme, & Klintsova, 2014; Gursky, Savage, & Klintsova, 2019; Hamilton, Boschen, Goodlett, Greenough, & Klintsova, 2012).

3.3|. Running distance

Previous work from our lab has demonstrated that running distance in WR animals does not significantly differ by postnatal treatment group (Helfer et al., 2009). Average running distance from PD 30–42 was 2.7 miles (±1.2 SD) per 24 hr for all postnatal treatment groups in both intervention groups. WR-only animals ran on average 8.5 miles (±3.8 SD) per 24 hr from PD 43–72, consistent with previously published work from our lab (Boschen et al., 2014, 2017; Hamilton et al., 2012). Animals were triple-housed in same-sex groups to avoid the negative outcomes of social isolation on behavioral and neuroanatomical parameters. Thus, the average running distance per individual rat could not be determined (Bennett et al., 1964; Diamond et al., 1964). A minimum of 10 days of free access to running wheels is sufficient to induce neuroanatomical changes (Snyder, Glover, Sanzone, Kamhi, & Cameron, 2009). Previous studies conducted in the lab demonstrate that both WR and “super-interventions” are sufficient to produce neuroanatomical alterations in our rodent model of FASD (Gursky & Klintsova, 2017; Hamilton et al., 2012, 2014).

3.4|. Cholinergic neuron number in NBM

A two-way ANOVA was conducted to compare the effects of postnatal treatment (SC, SI, or AE) and intervention group (SH, WR, or WREC) on the number of cholinergic neurons in NBM in adulthood. A main effect of postnatal treatment (F[2,38] = 4.689, p < .05, ηp2 = 0.20) was found. Tukey’s HSD post hoc test reveals a significant reduction in the number of ChAT+ neurons in NBM of AE (M = 1,317.94, SEM = ±158.89) compared to SC (M = 2,158.79, SEM = ±243.86) animals (p < .01). The relationship between the number of ChAT+ neurons in NBM of AE compared to SI animals is approaching the traditional threshold for statistical significance (p < .10). There was no main effect of intervention on cholinergic neuron number (F[2,38] = 2.05, p > .10, ηp2 = 0.10). The interaction between postnatal treatment x intervention group was not significant (p > .1, Figure 2b).

3.5|. Cholinergic neuronal volume in NBM

A two-way ANOVA was conducted to compare the effects of postnatal treatment and intervention group on the volume of ChAT+ neurons in NBM in adulthood. There was no significant main effect of postnatal treatment (F[2,38] = 0.47, p > .10, ηp2 = 0.04) or intervention (F[2,38] = 0.80, p > .10, ηp2 = 0.01) on cholinergic neuronal volume. The interaction between postnatal treatment × intervention group was not significant (p > .1, Figure 2c).

3.6 |. Structural volume of NBM

A two-way ANOVA was conducted to compare the effects of postnatal treatment and intervention group on the structural volume of NBM in adulthood. There was no significant main effect of postnatal treatment (F[2,38] = 0.83, p > .10, ηp2 = 0.04) or intervention (F[2,38] = 1.24, p > .10, ηp2 = 0.06) on the structural volume of NBM in adulthood. There was no significant interaction between effects of postnatal treatment x intervention group on total NBM volume (p > .1, Figure 2d).

3.7|. Cholinergic fiber number in Cg2

A two-way ANOVA was conducted to compare the effects of postnatal treatment and intervention group on the number of AChE+ fibers in Cg2 in adulthood. There was no significant main effect of postnatal treatment (F[2,38] = 0.51, p > .10, ηp2 = 0.03) or intervention (F[2,38] = 0.46, p > .10, ηp2 = 0.02) on fiber number in Cg2. The interaction between postnatal treatment x intervention group was not significant (p > .1, Figure 3b).

3.8|. Structural volume of Cg2

A two-way ANOVA was conducted to compare the effects of postnatal treatment and intervention group on the structural volume of Cg2 in adulthood. There was no significant main effect of postnatal treatment (F[2,38] = 0.50, p > .10, ηp2 = 0.03) or intervention (F[2,38] = 1.31, p > .10, ηp2 = 0.06) on the structural volume of Cg2. The interaction between postnatal treatment x intervention group was not significant (p > .1, Figure 3c).

4|. DISCUSSION

4.1|. Summary of major findings

Clinical and preclinical studies demonstrate that cholinergic neurotransmission and metabolism are particularly vulnerable to AE prior to and during the brain growth spurt. Behavioral deficits associated with FASD are partially mitigated by dietary choline supplementation and behavioral interventions that upregulate cholinergic neurotransmission. Specifically, PFC-dependent behaviors (e.g., executive function) are impaired by AE during the brain growth spurt. This histopathological study examined the impact of two behavioral interventions on the morphology of cortically-projecting cholinergic neurons in a rodent model of FASD. The data obtained reveal that cholinergic neurons in NBM, the primary source of cortical cholinergic innervation in the mammalian brain, are vulnerable to AE during the brain growth spurt, leading to a reduction in ChAT+ neuron number in NBM in adulthood. This effect is not mitigated by either behavioral intervention (WR or “super-intervention”) in adolescence–adulthood. Further, neither AE nor behavioral intervention altered the volume of ChAT+ neurons in NBM or the overall volume of NBM. Interestingly, neither AE nor behavioral intervention altered the number of AChE+ fibers in Cg2, the prelimbic sub-region of medial PFC, in adulthood.

4.2|. AE during the brain growth spurt reduces cholinergic neuron number in NBM in adulthood

Two possible explanations may account for the significant reduction in ChAT+ neuron number in adult NBM following AE during the brain growth spurt, or third-trimester equivalent. First, the teratogenicity of AE may result in neuron-specific apoptosis in NBM. The development of cholinergic neurons in rodents spans all pre- and postnatal stages with cholinergic neurogenesis occurring in early and mid-gestation preceding cholinergic fiber innervation of the cortex which occurs during the first few postnatal weeks (Gielow & Zaborszky, 2017; Kiss & Patel, 1992; Mechawar & Descarries, 2001; Meck et al., 2008). Cortical cholinergic innervation in humans is substantially matured by the second trimester of pregnancy (Candy et al., 1985). Brain cholinergic system maturation and metabolism are highly sensitive to neurotoxicity and environmental experience during the rodent perinatal and adolescent periods (Blusztajn, Slack, & Mellott, 2017; McKeon-O’Malley et al., 2003; Zeisel & Niculescu, 2006). Acetylcholine neurotransmission is necessary for capacities such as attention, learning, memory, and sleep, and, if disrupted by teratogenic exposure, could further perturb normative neurodevelopment. For example, choline insufficiency prevents neuronal and glial proliferation and differentiation and could significantly impede the brain growth spurt (Derbyshire & Obeid, 2020).

It is evident that prenatal AE disrupts natural choline metabolism in the brain, but the mechanism by which choline insufficiency causes secondary deficits in neuronal signaling, alterations to neuron morphology, and damages white matter tracts remains unknown. in vivo magnetic resonance spectroscopic imaging is a powerful tool used to measure neurometabolite levels, including the level of free choline-derived phospholipids. Alterations to free choline levels in the brain may indicate dysregulated cellular metabolism, cellular membrane breakdown, white matter tract damage, or changes to cholinergic enzymatic activity (ChAT or AChE). Preclinical and clinical studies indicate that the amount of free choline in the brain is altered following prenatal and early postnatal AE (Astley et al., 2009; Tang et al., 2019). Moreover, the severity of prenatal AE leads to targeted effects on different cell types in the developing brain. It has been reported that prenatal AE increases free choline levels in forebrain white matter tracts. However, evidence from a study involving nonhuman primates suggests that severe prenatal AE, producing pathology analogous to Fetal Alcohol Syndrome (FAS), leads to cholinergic neuronal apoptosis. Exogenous choline availability is a known rate-limiting step in acetylcholine synthesis. Wurtman et al. (1985) posited that significant choline insufficiency leads to cholinergic neuron auto-cannibalism, the mechanism by which cholinergic neurons breakdown portions of the cell membrane to release choline and upregulate acetylcholine synthesis. Indeed, it was demonstrated that free choline levels remain low in white matter regions, but increase in gray matter forebrain regions in nonhuman primates with severe prenatal AE (Astley, Weinberger, Shaw, Richards, & Clarren, 1995).

These findings suggest that cholinergic signaling may only be substantially altered in cases of severe prenatal AE. Further, prenatal choline supplementation may mitigate the adverse effects of prenatal AE on cognition by preventing apoptosis resulting from cholinergic neuronal auto-cannibalism and white matter tract damage. Given that the rodent model used in this study does not produce FAS-like pathology, it is unlikely that we observed cholinergic neuronal apoptosis. A comprehensive histological analysis of neonatal and adult rodent brain tissue samples (immediately following AE and in adulthood) is a logical future direction to examine neuron-specific apoptosis and its effect on the adult brain.

A second explanation for the observed reduction in the number of ChAT+ neurons following AE during the brain growth spurt postulates that neurons in NBM may have undergone a switch in neuronal phenotype. Rodent and human cholinergic neurons are susceptible to phenotypic alterations via neurotransmitter switching induced by disease pathology or environmental experiences such as exercise in adolescence and adulthood. Indeed, ChAT+ neurons in the peripheral and central nervous systems may become GABAergic or glutamatergic. It is suggested that cholinergic phenotypic plasticity is necessary for fine-tuning of movement and procedural learning across the lifespan as well as transitioning between physiological states like the “fight-or-flight” response and rest (Li & Spitzer, 2020; Spitzer, 2017). It is possible that AE during the brain growth spurt leads to a neurotransmitter switch, altering the phenotype of neurons in NBM in adulthood. Interestingly, our results suggest that cholinergic neurons in NBM are resistant to phenotypic change resulting from either behavioral intervention tested in adolescence–adulthood. Specific cholinergic nuclei are more amenable to phenotypic alteration in adolescence–adulthood than others, namely those in the midbrain, brainstem, and septum (Hall & Savage, 2016; Li & Spitzer, 2020; Spitzer, 2017). Phenotypic plasticity of cholinergic neurons in NBM is under explored. Previous studies examining ChAT+ neuron number and morphology in the basal forebrain in rodent models of FASD (AE on PD 4–10 or GD 1–22 [Moore, Lee, Paiva, Walker, & Heaton, 1998; Swanson, Tonjes, King, Walker, & Heaton, 1996], respectively) show that number and morphology of cholinergic neurons in other basal forebrain nuclei (medial septum) are resistant to the effects of AE during the brain growth spurt. Taken together, our findings indicate a unique sensitivity of cortically-projecting cholinergic neurons in NBM to early neurotoxicant exposure but not to environmental experience in adolescence–adulthood.

4.3|. AE during the brain growth spurt does not alter the number of cholinergic axons in prelimbic medial PFC in adulthood

Notably, despite the observed reduction in the number of ChAT+ neurons in NBM in adulthood, the number of cholinergic axons in the prelimbic PFC did not differ between AE and control groups and was not altered by either adolescent intervention (WR or “super-intervention”). Acetylcholine neurotransmission is necessary for experience-dependent plasticity in adulthood and is supported by cortical cholinergic innervation originating in the NBM (Baskerville, Schweitzer, & Herron, 1997). Cortical cholinergic innervation begins during the brain growth spurt and the density and laminar distribution of cholinergic fibers matures during adolescence in the rodent brain (Mechawar & Descarries, 2001). Thus, we predicted that the architecture of cholinergic fibers in medial PFC in the rodent brain would be significantly altered by either AE during the brain growth spurt and/or adolescent intervention.

The projection targets of cholinergic neurons in NBM are input-dependent in the rodent. For example, cholinergic neurons in NBM receiving input from the lateral septum and central amygdala project to the PFC, while cholinergic neurons in NBM receiving reciprocal input from somatosensory cortex, caudate putamen, and central amygdala project to the motor cortex (Gielow & Zaborszky, 2017). Further, human and rodent NBM are organized in a topographic manner such that rostral subregions of NBM project primarily to prelimbic medial PFC. Given what is known about the complexity of the organization of NBM, future studies would benefit from correlating the number of ChAT+ neurons in sub-regions of NBM with the number of projections in prelimbic medial PFC, specifically the most rostral sub-region (Bloem et al., 2014; Eckenstein et al., 1988; Mesulam & Geula, 1988).

The impact of the neuroanatomical observations described in the present study on cholinergic signaling are challenging to predict. Due to the complexity of cholinergic receptor localization and putative function, cortical cholinergic neurotransmission exerts both a direct excitatory and indirect inhibitory effect on cortical pyramidal cell function (Picciotto, Higley, & Mineur, 2012). Thus, while neither early postnatal AE nor either intervention changed the number of cholinergic projections in medial PFC in our study, it is possible that cholinergic signaling was affected. It is likely that the capacity for acetylcholine-dependent cortical plasticity was maintained despite teratogenic insult.

4.4|. Limitations

There exist several limitations to the current study that prevent comprehensive interpretation of the results. In particular, the relatively low sample size (n = 4–6 per postnatal treatment/intervention group) reduces the statistical power of the results and prevents comprehensive conclusion. Further, the exclusion of female rodents from this study limits translatability of the findings. Cholinergic enzymatic activity (namely AChE) is sexually dimorphic in the adolescent rodent striatum but not the cortex, however, AE during the brain growth spurt reduces AChE activity in the male brain such that it is comparable to that in the control female brain in early adolescence (Light et al., 1989).

4.5|. Conclusion and health policy implications

This study was the first to examine the impact of two behavioral interventions on cholinergic neuronal morphology in two prominent brain regions (NBM and PFC) in a rodent model of FASD. Findings from this study emphasize the impact of a binge AE during the rodent brain growth spurt on cholinergic neuron number in NBM in adulthood. Future studies will be necessary to investigate the impact of other known therapeutic strategies (i.e., choline supplementation) and further elucidate the mechanism by which alcohol perturbs cholinergic neuronal morphology and function in these brain regions.

Supplementary Material

Suppl Fig 1
Suppl Fig 2

ACKNOWLEDGMENTS

The research was supported by NIH/NIGMS COBRE: The Delaware Center for Neuroscience Research Grant 1P20GM103653-01A1 (Klintsova), NIH R21 AA026613-01 grant (Klintsova) and the NIH/NIAAA R01AA027269-01 (Klintsova). The authors thank Zachary Gursky, MS and Natalie Ginn for their work on data acquisition and assistance in training.

Funding information

National Institute of General Medical Sciences, Grant/Award Number: 1P20GM103653-01A1; National Institute on Alcohol Abuse and Alcoholism, Grant/Award Numbers: R01 AA027269-01 (AK), R21 AA026613-01 grant (AK)

Footnotes

SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of this article.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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

Supplementary Materials

Suppl Fig 1
NIHMS1718236-supplement-Suppl_Fig_1.tif (981.3KB, tif)
Suppl Fig 2
NIHMS1718236-supplement-Suppl_Fig_2.tif (7.6MB, tif)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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