Type 3 Adenylyl Cyclase and Somatostatin Receptor 3 Expression Persists in Aged Rat Neocortical and Hippocampal Neuronal Cilia

Abstract

The primary cilia of forebrain neurons assemble around birth and become enriched with neuromodulatory receptors. Our understanding of the permanence of these structures and their associated signaling pathways in the aging brain is poor, but they are worthy of investigation because disruptions in neuronal cilia signaling have been implicated in changes in learning and memory, depression-like symptoms, and sleep anomalies. Here, we asked whether neurons in aged forebrain retain primary cilia and whether the staining characteristics of aged cilia for type 3 adenylyl cyclase (ACIII), somatostatin receptor 3 (SSTR3), and pericentrin resemble those of cilia in younger forebrain. To test this, we analyzed immunostained sections of forebrain tissues taken from young and aged male Fischer 344 (F344) and F344 × Brown Norway (F344 × BN) rats. Analyses of ACIII and SSTR3 in young and aged cortices of both strains of rats revealed that the staining patterns in the neocortex and hippocampus were comparable. Virtually every NeuN positive cell examined possessed an ACIII positive cilium. The lengths of ACIII positive cilia in neocortex were similar between young and aged for both strains, whereas in F344 × BN hippocampus, the cilia lengths increased with age in CA1 and CA3, but not in dentate gyrus (DG). Additionally, the percentages of ACIII positive cilia that were also SSTR3 positive did not differ between young and aged tissues in either strain. We also found that pericentrin, a protein that localizes to the basal bodies of neuronal cilia and functions in primary cilia assembly, persisted in aged cortical neurons of both rat strains. Collectively, our data show that neurons in aged rat forebrain possess primary cilia and that these cilia, like those present in younger brain, continue to localize ACIII, SSTR3, and pericentrin. Further studies will be required to determine if the function and signaling pathways regulated by cilia are similar in aged compared to young brain.

Keywords: G protein-coupled receptor; aging; cerebral cortex; cilium.

Figure 1

Type 3 adenylyl cyclase (ACIII)-positive cilia in young and aged rat forebrain. (A–D) Confocal images of 2 months (mos) (left panels) and 34 mos (right panels) Fischer F344 (F344) × Brown Norway (BN) rat layer 2/3 of neocortex (Nctx) (A,B) and CA1 region of hippocampus (C,D) immunostained for ACIII (green) and NeuN (red). Nuclei were stained with DAPI. (E) The percentage of NeuN positive cells also possessing ACIII positive cilia. (F,G) The lengths of ACIII positive cilia in the Nctx (F) and hippocampal subregions (G) of juvenile (N = 4) and aged (N = 4) rat brains. Data was statistically analyzed using Student’s t-test. *p < 0.05. (H,I) Confocal images of 6 and 24 mos F344 rat layer 2/3 of frontal neocortex immunostained for ACIII (green) and NeuN (red). Nuclei were stained with DAPI. (J) The lengths of ACIII positive cilia in 6 and 24 mos F344 Nctx. Scale bars in B,D,I = 10 μm.

Figure 2

Somatostatin receptor 3 (SSTR3)-positive cilia in young and aged rat forebrain. (A–F) Epifluorescent images of 2 and 34 mos F344 × BN layer 2/3 primary motor neocortex (M1) co-immunolabeled for ACIII (green), SSTR3 (red) and NeuN (blue). Arrowheads indicate cilia positive for both SSTR3 and ACIII. (G–J) Percent of NeuN+ neurons that possess SSTR3+ cilia in M1 (G) and hippocampal DG (H), CA1 (I) and CA3 (J) of 2 and 34 mos F344 × BN rats. (K–P) Images of 6 (left panels) and 24 mos (right panels) F344 rat layer 2/3 frontal neocortex immunostained for SSTR3 (red), ACIII (green) and NeuN (red). The arrowheads indicate cilia positive for both SSTR3 and ACIII, while the arrows (A,C,E,L,N,P) show examples of an ACIII positive cilium that is SSTR3 negative. (Q) Percent of NeuN positive neurons that possess SSTR3 positive cilia in frontal neocortex. Scale bars (A,O) = 10 μm.

Figure 3

Pericentrin localizes to the base of neuronal SSTR3+ cilia in young and aged rat cortex. Sections of F344 × BN (A) and F344 (B) rat frontal cortex were immunostained for pericentrin (PCNT, green), SSTR3 (red), and NeuN (blue). Confocal images show PCNT at the base (arrows) of SSTR3+ cilia (arrowheads) in layer 2/3 neurons of both young (left panels) and aged (right panels) tissues. *lipofuscin. Scale bar = 10 μm.