Age-related changes in basal forebrain afferent activation in response to food paired stimuli

Abstract

The basal forebrain contains a phenotypically-diverse assembly of neurons, including those using acetylcholine as their neurotransmitter. This basal forebrain cholinergic system projects to the entire neocortical mantle as well as subcortical limbic structures that include the hippocampus and amygdala. Basal forebrain pathology, including cholinergic dysfunction, is thought to underlie the cognitive impairments associated with several age-related neurodegenerative conditions, including Alzheimer's disease. Basal forebrain dysfunction may stem, in part, from a failure of normal afferent regulation of cholinergic and other neurons in this area. However, little is understood regarding how aging, alone, affects the functional regulation of basal forebrain afferents in the context of motivated behavior. Here, we used neuronal tract-tracing combined with motivationally salient stimuli in an aged rodent model to examine how aging alters activity in basal forebrain inputs arising from several cortical, limbic and brainstem structures. Young rats showed greater stimulus-associated activation of basal forebrain inputs arising from prelimbic cortex, nucleus accumbens and the ventral tegmental area compared with aged rats. Aged rats also showed increased latency to respond to palatable food presentation compared to young animals. Changes in activation of intrinsic basal forebrain cell populations or afferents were also observed as a function of age or experimental condition. These data further demonstrate that age-related changes in basal forebrain activation and related behavioral and cognitive functions reflect a failure of afferent regulation of this important brain region.

Keywords: Aging; Basal forebrain; Cholinergic; Feeding; Motivated behavior; Tract-tracing.

Fig. 1.. Feeding response latency in young and old rats.

Change in response latency (time to approach and consume the food) of young (black circle) and old (black square) rats across a 7 day period of daily exposure to the food-paired stimulus. Significant age-related differences emerged on day 3 and persisted across the training schedule, with old rats slower to respond. *p < 0.05 old vs young rats.

Fig. 2.. Retrograde labeling following basal forebrain CTb deposits.

(A) Representative CTb deposit site in ventral pallidum (VP), corresponding approximately to the region outlined by the box in the lower left diagram of panel B. Brown labeling indicates immunoreactivity for CTb around the deposit site. CPu, caudate-putamen; GP, globus pallidus. (B) Composite showing approximate regions where cell counts were performed. See text for further detail. Figures in panel B adapted from the atlas of Paxinos and Watson [18]. (C) CTb density (cells/mm²) of basal forebrain afferent regions including prelimbic cortex (PLC), infralimbic cortex (ILC), insular cortex (IC), lateral hypothalamus (LH), claustrum (Cl), nucleus accumbens (NuccAcc), central amygdala (CeA), and ventral tegmental area (VTA). Young and aged animals had equivalent numbers of retrogradely-labeled neurons in all brain regions sampled.

Fig. 3.. Phenotypic profile of activated basal forebrain neurons.

(A) Parvalbumin (PV)-positive, putatively GABAergic, basal forebrain neurons showed significantly greater activation in response to the food-paired stimulus (E1 condition) in young vs old rats. (B,C) Representative images of cFos/PV + double labeling (black arrows) in the basal forebrain of a young (B) or old (C) rat after the food paired stimulus. (D) ChAT-positive (cholinergic) basal forebrain neurons showed neither age- nor experimental condition-dependent activation. (E,F) Representative images of cFos/ChAT double labeling (black arrows) in the basal forebrain in a young (E) or old (F) rat. (G) vGluT1-positive (putatively glutamatergic) basal forebrain neurons showed neither age- nor experimental condition-dependent activation. (H,I) Double labeled (black arrow) cFos/vGluT in the basal forebrain of young (H) or old (I) rat. Insets in C, F and I show high-magnification (40x) representative samples of double-labeled neurons. *p < 0.05. Scale bar (I), 50 μm.

Fig. 4.. Activation of basal forebrain afferents from cortex.

(A) Young E1 rats showed increased activation of basal forebrain afferents originating from prelimbic cortex relative to both control conditions and relative to aged E1 rats. Aged rats showed no treatment-related differences in percent of CTb-positive neurons that were also positive for cFos. (D) Main effects of treatment group were seen in both young and aged rats in activation of basal forebrain afferents originating from the infralimbic cortex, with E1 rats showing less activation than both control conditions. (G) No effects of age or treatment group on cFos expression were seen in retrogradely-labeled insular cortex neurons. (B)(E)(H) Representative photomicrographs of cFos/CTb double labeling in the PLC, ILC, and IC, respectively, in young rats after the food paired stimulus (E1). (C)(F)(I) Representative photomicrographs of cFos/CTb double labeling in the PLC, ILC, and IC, respectively, in old rats after the food paired stimulus (E1). Black arrows indicate double-labeled neurons. Insets in C, F and I show higher-magnification (40x) representative samples of double-labeled neurons. *p < 0.05, **p < 0.01. scale bar, 100 μm.

Fig. 5.. Activation of claustrum or central amygdala projections to basal forebrain.

(A) There were no significant treatment- or age-related differences in activation of claustrum projections to basal forebrain. (D) In central amygdala, retrogradely-labeled neurons showed significantly less activation in the C2 control group vs the C1 control group in young rats. No differences were noted in aged rats. (B)(E) Representative photomicrographs of cFos/CTb double labeling (black arrow) in the claustrum and central amygdala in young rats after the food paired stimulus. (C)(F) Representative photomicrographs from claustrum (C) or central amygdala (F) of an old rat image after the food paired stimulus. Black arrows indicate double-labeled neurons. *p < 0.05. scale bar, 100 μm.

Fig. 6.. Activation of other limbic-related projections to basal forebrain.

(A) There were no significant differences between young and old rats in activation of lateral hypothalamic projections to basal forebrain in any experimental condition. (D) Both young and old rats showed significant activation of basal forebrain afferents from the nucleus accumbens in response to the food-paired stimulus (E1 condition). However, the magnitude of this increase was significantly larger in young animals. (G) The food-paired stimulus (E1) produced robust activation of VTA projections to basal forebrain in young rats versus both control conditions. No such response was observed in old rats. (B)(E)(H) Representative images of cFos/CTb double labeling (black arrows) in the LH, NAcc, and VTA, respectively, in young rats after the food paired stimulus (E1). (C)(F)(I) Representative images of cFos/CTb double labeling (black arrows) in the LH, NAcc, and VTA, respectively, in old rats after the food paired stimulus (E1). Black arrows indicate double-labeled neurons. *p < 0.05, **p < 0.01. scale bar, 100 μm.

Fig. 7.. Activation of VTA dopamine neurons.

(A)(B) Representative photomicrographs of double-labeling for tyrosine hydroxylase (TH; brown) and c-Fos (black) in the VTA of young (A) & aged (B) rats rats from the E1 group. Arrows indicate examples of double-labeled cells. (C) There was no significant effect of age or experimental condition on the percentage of dopaminergic neurons expression c-Fos, although there was a trend for aged animals in the E1 group to have fewer double-labeled cells than their young counterparts. scale bar, 100 μm.