Research reportMuscarinic receptor/G-protein coupling is reduced in the dorsomedial striatum of cognitively impaired aged rats
Highlights
► Cognitive flexibility becomes impaired by old age. ► Muscarinic receptor stimulation of GTP binding, a measure of receptor coupling, is blunted in the dorsal striatum of aged rats. ► Muscarinic receptor levels remain stable in the dorsal striatum of aged rats. ► Data indicate that muscarinic receptor function but not level contributes to increased cognitive rigidity in old age.
Introduction
Under the umbrella term of executive function is the flexible use of knowledge, termed here as behavioral flexibility [1]. Behavioral flexibility is impaired in aged primates [2], dogs [3] and rodents [4]. Although the neurobiological mechanism underlying the decline in behavioral flexibility in the aged brain is unknown, pharmacological and lesion studies in adult rats indicate that fronto-striatal function is critical. In adult rats, impairments in reversal learning occur after lesions or inactivation of the orbito-frontal region of the prefrontal cortex or the dorsomedial region of the striatum [5], [6], [7]. However, based on the anatomical connections between the orbito-frontal cortex and the dorsomedial striatum [1], [8], [9], [10], mechanistic dysfunction in either or both of these regions have the potential to result in an age-related decline in behavioral flexibility and increase in cognitive rigidity.
In the striatum, age-related declines in striatal acetylcholine synthesis, release and degradation have been identified [11], [12], [13], [14]. Additionally, pharmacological antagonism of striatal muscarinic acetylcholine receptors with pirenzepine, a muscarinic receptor antagonist, impairs behavioral flexibility (reversal learning) when infused into the dorsomedial striatum of adult rats [15]. However, it is not known if age-related alterations in striatal cholinergic signaling are associated with the decrease in behavioral flexibility observed in aging.
The predominate muscarinic receptor subtypes in the striatum are M1, M2 and M4 (reviewed in [16]). M2/M4 AChRs are located on the large, aspiny striatal cholinergic interneurons, also called tonically active neurons (TANS). TANS make up about 2% of all striatal neurons and have a major modulatory affect on striatal neurotransmission, particularly in regard to dopaminergic regulation (see [16]. Because of the major influence of the TANS on striatal neurotransmission, we focused our study on the M2/M4 muscarinic receptor subtypes.
M2/M4 receptor function and receptor density was analyzed in the striatum from behaviorally characterized young, middle-aged and aged rats. All rats were tested in an attentional set-shifting task and in the Morris water maze to assess fronto-striatal and hippocampal function, respectively. M2/M4 AChRs were assessed for both the function (receptor/G-protein coupling) and level. G-protein-coupled receptor function can be assessed by the ability of agonist to activate the binding of GTP to the alpha subunit of the G-protein complex. M2/M4 muscarinic receptors were stimulated with the agonist oxotremorine-M [17], [18]. Receptor stimulation by oxotremorine-M activates the G-protein Gαq11 and the bound GDP (“off state”) is replaced with GTP (“on state”). The GTP is radiolabeled (GTPγS35) and non-hydrolysable, allowing for quantification using an autoradiographic method [19]. To measure M2/M4 receptor levels, the muscarinic receptor antagonist [3H]AFDX-398 was used. It preferentially binds to the M2 and M4 AChRs at the concentration used in this study (2 nM) ([18], [20], [21]). Muscarinic receptor function and levels were quantified in the same brain regions of the same rats and data were analyzed relative to both age and cognitive ability.
Section snippets
Subjects
Male Fisher 344 × Brown Norway (F344BNF1, NIA-Harlan, Chicago, IL) rats were all of the same cohort with all ages arriving in the same shipment. Rats were housed individually once they arrived at the laboratory for use as subjects. Rats were on a 12-h light cycle and food and water were available ad lib unless otherwise indicated. There were three age groups: Young (N = 7), middle-aged (N = 7) and aged (N = 7). Rats were assessed at 8, 18 and 27 months of age for spatial learning in the water maze
Attentional set-shifting
Behavioral flexibility was assessed using an attentional set-shifting discrimination paradigm that assesses both reversal learning and set shifting. Fig. 1 shows the number of training trials needed to reach criterion for all phases of the attentional set-shifting task. A main effect of age (Phase, F(2,18) = 5.07, p < 0.05) indicates that the aged group required significantly more trials to reach criterion than the young and middle-aged groups and that the young and middle-aged groups did not
Discussion
The experiments described here assessed the integrity of the cholinergic system of the striatum in rats at 6, 18, and 24 months of age relative to cognitive performance on an attentional set-shifting and spatial learning task. The results of the cognitive assessment showed that spatial learning ability was impaired by middle-age whereas cognitive flexibility remained intact until very old age. Additionally, the pattern of results for oxotremorine-M-stimulated [35S]GTPγS binding were similar to
Conclusions
In this study, normal, healthy aged rats demonstrated an age-related increase in cognitive rigidity, a measure of executive function. Cognitive rigidity was associated with impaired muscarinic receptor signaling in the dorsomedial striatum. These results are consistent with lesion and pharmacological studies in adult rats that demonstrate impaired reversal learning as a consequence of dorsomedial striatal damage or cholinergic antagonism. Our data indicates that neurobiological changes in the
Acknowledgements
The authors wish to thank Adam Wilson, Tammy Sexton, Hilary Smith and Ruoyu Xiao for their expert technical assistance. Funding was provided by NIA AG-020572 (MMN), NIA P01 AG-011370 (WES, MMN, ENM) and NIDA DA-06634 (SRC).
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2021, NeuropharmacologyCitation Excerpt :In rodents, cognitive flexibility depends on the mPFC and may be evaluated through set-shifting tasks implemented across a variety of modalities and experimental settings (Birrell and Brown, 2000; Floresco et al., 2008; Ragozzino et al., 1999). While decline of hippocampus-dependent spatial memory is also a reliable feature of rodent brain aging (Barnes et al., 1997; Bizon et al., 2009; Gallagher et al., 1993; McQuail and Nicolle, 2015), individual differences in set-shift performance do not correlate with hippocampal-dependent learning and memory as evaluated in the Morris water maze (Barense et al., 2002; Beas et al., 2013; Nieves-Martinez et al., 2012). Further, analysis of errors committed by aged rats during the set-shift phase of the task reveals that slower acquisition of the new rule is due to perseverative errors made in response to the previously reinforced stimulus (i.e. location of the illuminated light; Beas et al., 2013, 2017; and see also Beas et al., 2016), mirroring the pattern of behavioral deficits produced by mPFC inactivation (Floresco et al., 2008).
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