Maternal choline supplementation improves spatial mapping and increases basal forebrain cholinergic neuron number and size in aged Ts65Dn mice

Abstract

Down syndrome (DS) is marked by intellectual disability (ID) and early-onset of Alzheimer's disease (AD) neuropathology, including basal forebrain cholinergic neuron (BFCN) degeneration. The present study tested the hypothesis that maternal choline supplementation (MCS) improves spatial mapping and protects against BFCN degeneration in the Ts65Dn mouse model of DS and AD. During pregnancy and lactation, dams were assigned to either a choline sufficient (1.1g/kg choline chloride) or choline supplemented (5.0g/kg choline chloride) diet. Between 13 and 17months of age, offspring were tested in the radial arm water maze (RAWM) to examine spatial mapping followed by unbiased quantitative morphometry of BFCNs. Spatial mapping was significantly impaired in unsupplemented Ts65Dn mice relative to normal disomic (2N) littermates. Additionally, a significantly lower number and density of medial septum (MS) hippocampal projection BFCNs was also found in unsupplemented Ts65Dn mice. Notably, MCS significantly improved spatial mapping and increased number, density, and size of MS BFCNs in Ts65Dn offspring. Moreover, the density and number of MS BFCNs correlated significantly with spatial memory proficiency, providing support for a functional relationship between these behavioral and morphometric effects of MCS for trisomic offspring. Thus, increasing maternal choline intake during pregnancy may represent a safe and effective treatment approach for expectant mothers carrying a DS fetus, as well as a possible means of BFCN neuroprotection during aging for the population at large.

Keywords: Alzheimer's disease; Down syndrome; Hippocampus; Medial septum; Nucleus basalis; Radial arm water maze; Spatial memory; Stereology; Vertical limb of the diagonal band.

Fig. 1. RAWM performance

(A) Average errors per trial (collapsed across sessions) in the HP Task was significantly higher for the unsupplemented Ts65Dn mice than their 2N counterparts. MCS significantly improved performance for Ts65Dn (p = 0.011) but not 2N mice; (B) Mean errors per trial across the 15 sessions of the HP task; (C) Mean errors in the VP task across the 6 sessions on this task; no significant groups differences were seen. Abbreviations: 2N Ch: disomic mice born to dams maintained on a choline normal diet (n = 19); Ts Ch: Ts65Dn mice born to dams maintained on a choline normal diet (n = 13); 2N Ch+: disomic mice born to dams maintained on a choline supplemented diet (n = 20); Ts Ch+: Ts65Dn mice born to dams maintained on a choline supplemented diet (n = 21); HP: Hidden platform task of the radial arm watermaze; VP: Visible platform task of the radial arm watermaze; MCS: maternal choline supplementation; * p ≤ 0.05, ** p ≤ 0.001.

Fig. 2. Density of ChAT-, p75^NTR-, and TrkA-immunoreactive cells in the basal forebrain

(A) In the MS, Ts Ch mice showed a significantly lower ChAT-immunoreactive density relative to 2N Ch mice (p = 0.008), whereas both groups of Ts65Dn mice showed reduced density of p75^NTR-immunoreactive and TrkA-immunoreactive neurons relative to 2N mice (p75^NTR: p = 0.0001; TrkA: p = 0.002). MCS significantly increased the density of ChAT-immunoreactive neurons in Ts65Dn mice (p = 0.036); (B) No significant differences were seen in the VDB; (C) In the NBM/SI, both groups of Ts65Dn mice showed reduced p75^NTR-immunoreactive neuron density relative to 2N mice (p < 0.0001). Abbreviations: 2N Ch: disomic mice born to dams maintained on a choline normal diet; Ts Ch: Ts65Dn mice born to dams maintained on a choline normal diet; 2N Ch+: disomic mice born to dams maintained on a choline supplemented diet; Ts Ch+: Ts65Dn mice born to dams maintained on a choline supplemented diet; Geno: Main effect of Genotype; MS: Medial Septum; VDB: Ventral Diagonal Band; NBM/SI: Nucleus Basalis of Meynert/Substantia Innominata; BFCNs: basal forebrain cholinergic neurons; ChAT: Choline Acetyltransferase; p75^NTR: pan neurotrophin receptor; TrkA: tyrosine kinase A receptor; MCS: maternal choline supplementation;*p ≤ 0.05, **p ≤ 0.001.

Fig. 3. Number of ChAT-, p75^NTR-, and TrkA-immunoreactive neurons in the basal forebrain

(A) In the MS, MCS significantly increased the number of ChAT-immunoreactive neurons in Ts65Dn mice (p = 0.009); (B) In the VDB, both groups of Ts65Dn mice showed a significantly higher number of TrkA-immunoreactive neurons (p = 0.036). MCS increased the number of ChAT-immunoreactive BFCNs for both genotypes (p = 0.021); (C) In the NBM/SI, Ts65Dn mice exhibited a significantly higher number of p75^NTR-immunoreactive neurons than the 2N mice (p < 0.0001). Abbreviations: 2N Ch: disomic mice born to dams maintained on a choline normal diet; Ts Ch: Ts65Dn mice born to dams maintained on a choline normal diet; 2N Ch+: disomic mice born to dams maintained on a choline supplemented diet; Ts Ch+: Ts65Dn mice born to dams maintained on a choline supplemented diet; Geno: Main effect of Genotype; MS: Medial Septum; VDB: Ventral Diagonal Band; NBM/SI: Nucleus Basalis of Meynert/Substantia Innominata; BFCNs: basal forebrain cholinergic neurons; ChAT: Choline Acetyltransferase; p75^NTR: pan neurotrophin receptor; TrkA: tyrosine kinase A receptor; MCS: maternal choline supplementation; * p ≤ 0.05, ** p ≤ 0.001.

Fig. 4. Size of ChAT-, p75^NTR-, and TrkA-immunoreactive cells in the basal forebrain

(A) In the MS, MCS increased TrkA-immunoreactive BFCNs for both genotypes (p = 0.050); There was no effect of genotype, maternal diet, nor their interaction in (B) the VDB, or (C) the NBM/SI. Abbreviations: 2N Ch: dis-omic mice born to dams maintained on a choline normal diet; Ts Ch: Ts65Dn mice born to dams maintained on a choline normal diet; 2N Ch+: disomic mice born to dams maintained on a choline supplemented diet; Ts Ch+: Ts65Dn mice born to dams maintained on a choline supplemented diet; MS: Medial Septum; VDB: Ventral Diagonal Band; NBM/SI: Nucleus Basalis of Meynert/Substantia Innominata; BFCNs: basal forebrain cholinergic neurons; ChAT: Choline Acetyltransferase; p75^NTR: pan neurotrophin receptor; TrkA: tyrosine kinase A receptor; MCS: maternal choline supplementation; * p ≤ 0.05, ** p ≤ 0.001.

Fig. 5. Regional basal forebrain areas

(A) MCS decreased MS area for 2N mice (p = 0.046), but had no effect for Ts65Dn mice; (B) Unsupplemented Ts65Dn mice showed significantly larger VDB area than unsupplemented 2N mice (p = 0.016), a pattern also seen for the (C) NBM/SI (p = 0.033). MCS had no effect on VDB or NBM/SI for either genotype. Abbreviations: 2N Ch: disomic mice born to dams maintained on a choline normal diet; Ts Ch: Ts65Dn mice born to dams maintained on a choline normal diet; 2N Ch+: disomic mice born to dams maintained on a choline supplemented diet; Ts Ch+: Ts65Dn mice born to dams maintained on a choline supplemented diet; MS: Medial Septum; VDB: Ventral Diagonal Band; NBM/SI: Nucleus Basalis of Meynert/Substantia Innominata; BFCNs: basal forebrain cholinergic neurons; ChAT: Choline Acetyltransferase; p75^NTR: pan neurotrophin receptor; TrkA: tyrosine kinase A receptor; MCS: maternal choline supplementation; * p ≤ 0.05, ** p ≤ 0.001.

Fig. 6. Correlations between HP performance and BFCN indices in the MS

Mean errors in block 3 of the HP task was negatively correlated with (A) Density of ChAT-immunoreactive BFCNs (p = 0.014), (B) Density of TrkA-immunoreactive BFCNs (p = 0.095); (C) Cell size of p75^NTR-immunoreactive BFCNs (p = 0.036), and (D) Number of p75^NTR- immunoreactive BFCNs (p = 0.062). Abbreviations: 2N Ch: disomic mice born to dams maintained on a choline normal diet; Ts Ch: Ts65Dn mice born to dams maintained on a choline normal diet; 2N Ch+: disomic mice born to dams maintained on a choline supplemented diet; Ts Ch+: Ts65Dn mice born to dams maintained on a choline supplemented diet; HP: Hidden platform task of the radial arm watermaze; BFCNs: basal forebrain cholinergic neurons; ChAT: Choline Acetyltransferase; p75^NTR: pan neurotrophin receptor; TrkA: tyrosine kinase A receptor.