ArticlesSubunit and region-specific decreases in nicotinic acetylcholine receptor mRNA in the aged rat brain
Introduction
An impairment in cholinergic systems is a highly consistent finding in human dementia [3], [4], [6] and is thought to contribute to the cognitive deficits observed in this pathology. Among cholinergic markers, marked decreases in high affinity binding for nicotinic agonists have been most consistently observed in the telencephalic regions of demented patients [22], [43], [50], [67], [68]. It has been proposed that the nicotinic deficit in demented patients contributes to their cognitive impairment. This hypothesis is based on the evidence that nicotine can improve memory performance and learning in rodents and non-human primates and vigilance and rapid information processing in humans [32]. Accordingly, nicotinic receptor blockers (e.g., mecamylamine) impair performance in spatial memory and other cognitive tasks [19], [33], [39]. Notably, nicotine is particularly effective in recovering cognitive deficits caused by lesions of the cholinergic system in animals and ameliorating cognitive functions in both aged humans and animals [28], [63].
Several data are available on changes in cholinergic systems in normal aging in humans [18]. Markers of the telencephalic cholinergic terminals, such as choline acetyltransferase (ChAT) activity and acetylcholine (ACh) synthesis, are substantially unchanged in aged rodents, whereas impaired stimulation induced ACh release has been reported by several groups [26], [40], [47], [48]. Age-related changes in cholinergic receptors, both muscarinic and nicotinic, in rodent brain have been inconsistently observed and decreases, when present, are modest [16], [22], [40]. On the other hand, there are several reports on decreases in electrical responsivity to cholinergic substances in aged rodent cortex and hippocampus [54], [61], [62]. However, the vast majority of the studies has been oriented towards assessment of muscarinic effects, whereas little is known on age-related changes in the effects of nicotinic substances.
Neuronal nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of ligand-gated ion channels. They are membrane proteins composed of five subunits which are distinguished in subunits carrying the principal component of the ACh binding site (named α2–α9) and subunits carrying the complementary component of the ACh binding site (named non-α or β2–β4) [12], [30], [34], [65]. Based on structural, pharmacological and phylogenetic analysis, neuronal subunits have been subdivided into two subfamilies, the α-bungarotoxin-sensitive and the α-bungarotoxin-insensitive. The first subfamily is composed of α7–α8 subunits, which can form functional homo-oligomers in reconstituted systems [9], [17], [57]. The second subfamily is composed of α2–α6 and β2–β4 subunits. With the exception of β3, whose contribution to nAChRs is still unknown [31, but see 23], these subunits can form a number of functionally different hetero-oligomers composed of two, three or four subunits [14], [15], [24], [35], [36], [56], [64], [66]. It is therefore likely that several nAChR isoforms exist in the mammalian brain. Accordingly, electrophysiological studies in brain and ganglionic preparations have revealed nAChRs with distinct pharmacological specificities and ion channel properties, and several different combinations of nAChR subunit mRNAs and/or proteins have been identified in central and peripheral nervous structures [58].
However, available evidence indicates that only a few combinations exist in large amount in the rodent brain. α4 and β2 subunits, which are known to form the nAChR isotype with high affinity for nicotine [21], [52], constitute more than 90% of α-bungarotoxin-insensitive nAChRs in the CNS. α3 and β4, which form the main nAChR responsible for nicotinic synaptic transmission in autonomic ganglia [15], are highly enriched in the medial habenula [31], [65]. α6 and β3 have not yet been shown to form functional nAChR when coexpressed in reconstituted systems; however, they are highly and selectively enriched in catecholaminergic nuclei, especially the dopaminergic mesostriatal neurons and the noradrenergic neurons of the locus coeruleus [31]. It has therefore been proposed that they can form, together with other α and/or β subunits (such as β2, [53]), the nAChR responsible for catecholamine release in basal ganglia and hippocampus [31].
In this study, we have analyzed the possible age-related changes in the mRNA levels for the α and β nAChR subunits constituting the major putative nAChR isoforms (α4–β2, α3–β4, α6–β3, α7, see above) in 7- to 32-month-old rats. These changes have been correlated with the level of binding for ligands selective for the α-bungarotoxin sensitive (125I-α-bungarotoxin, [11]) and insensitive (3H-epibatidine,5,55) subfamilies. High affinity epibatidine binding in the CNS corresponds to high affinity nicotine binding, i.e. to nAChRs formed by α4 and β2 [[72], [73]] in the vast majority of tel- and diencephalic areas. One exception is the medial habenula, where epibatidine binding mainly corresponds to nAChRs which do not contain β2 but most likely α3–β4 [[72], [73]].
Section snippets
Animals
Twelve-month-old male Sprague–Dawley rats (Charles River, Italy) were housed in a specific pathogen–free (SPF) area at Sigma-Tau S.p.A. laboratories (Pomezia Terme, Italy). Standard monitoring of this area did not reveal any infectious event during the study period. Animals were housed three per cage and received tap water and standard laboratory diet ad libitum (4RF18, Mucedola S.r.L. Italy), which contained a minimum of 16% protein, 2.5% fat, and a maximum of 7.5% fiber. The animal rooms were
Age-related changes in nicotinic receptor mRNA levels
Overall, at both telencephalic and diencephalic levels, α4 mRNA levels progressively decreased from adulthood (7–14 months of age) to senescence (29–32 months of age) Fig. 1, Fig. 2. In cortical and subcortical telencephalic areas a slight increase in α4 mRNA levels was detected from 7 to 14 months of age. The age-related decrease was relatively constant in different areas, reaching at 29 months of age around 70–80% of adult values. In some areas (see, e.g., the thalamus), a further decrease
Discussion
The density of nicotinic binding sites has been reported to decrease markedly in Alzheimer’s patients [22], [43], [50], [67], [68]. Instead, only small and inconsistent decreases are observed in both humans and rodents during normal aging [16], [18], [22], [40]. In agreement with previous findings, this paper shows that equilibrium binding of 3H-epibatidine (a ligand selective for the α-bungarotoxin-insensitive subfamily of neuronal nAChR subunits) does not change significantly from 7 to 32
Acknowledgements
We thank Dr. O. Ghirardi and Sigma Tau (Pomezia Terme, Italy) for their generous gift of the animals used in these experiments. This work has been supported by grants from Italian CNR and MURST.
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