Nicotinic Receptor Subunit Distribution in Auditory Cortex: Impact of Aging on Receptor Number and Function

Abstract

The presence of novel or degraded communication sounds likely results in activation of basal forebrain cholinergic neurons increasing release of ACh onto presynaptic and postsynaptic nAChRs in primary auditory cortex (A1). nAChR subtypes include high-affinity heteromeric nAChRs commonly composed of α4 and β2 subunits and low-affinity homomeric nAChRs composed of α7 subunits. In young male FBN rats, we detail the following: (1) the distribution/expression of nAChR subunit transcripts in excitatory (VGluT1) and inhibitory (VGAT) neurons across A1 layers; (2) heteromeric nAChR binding across A1 layers; and (3) nAChR excitability in A1 layer (L) 5 cells. In aged rats, we detailed the impact of aging on A1 nAChR subunit expression across layers, heteromeric nAChR receptor binding, and nAChR excitability of A1 L5 cells. A majority of A1 cells coexpressed transcripts for β2 and α4 with or without α7, while dispersed subpopulations expressed β2 and α7 or α7 alone. nAChR subunit transcripts were expressed in young excitatory and inhibitory neurons across L2-L6. Transcript abundance varied across layers, and was highest for β2 and α4. Significant age-related decreases in nAChR subunit transcript expression (message) and receptor binding (protein) were observed in L2-6, most pronounced in infragranular layers. In vitro patch-clamp recordings from L5B pyramidal output neurons showed age-related nAChR subunit-selective reductions in postsynaptic responses to ACh. Age-related losses of nAChR subunits likely impact ways in which A1 neurons respond to ACh release. While the elderly require additional resources to disambiguate degraded speech codes, resources mediated by nAChRs may be compromised with aging.SIGNIFICANCE STATEMENT When attention is required, cholinergic basal forebrain neurons may trigger increased release of ACh onto auditory neurons in primary auditory cortex (A1). Laminar and phenotypic differences in neuronal nAChR expression determine ways in which A1 neurons respond to release of ACh in challenging acoustic environments. This study detailed the distribution and expression of nAChR subunit transcript and protein across A1 layers in young and aged rats. Results showed a differential distribution of nAChR subunits across A1 layers. Age-related decreases in transcript/protein expression were reflected in age-related subunit specific functional loss of nAChR signaling to ACh application in A1 layer 5. Together, these findings could reflect the age-related decline in selective attention observed in the elderly.

Keywords: ACh aging; auditory cortex; nAChR distribution.

Figure 1.

nAChR subunit transcript expression in auditory cortex area A1. A–C, Low power images of β2, α4, and α7 transcript expression (red dots). Blue represents hematoxylin counterstain. D–F, Plots of cells (circular symbols) containing transcripts of each subunit. Symbol shading represents transcript density (1–2, 3–5, 6–10, >10 transcripts/cell). Laminar boundaries indicated in transparent vertical bars. wm, White matter; CA2, cornu ammonis region 2 of hippocampus. Scale bar, 250 µm.

Figure 2.

Transcript coexpression controls, FISH assays. A, Positive controls: UBC (yellow dots), Polr2a (red dots), Ppib (aqua dots). B, Negative control: dapB (all color channels). Light gray represents DAPI. Scale bars, 10 µm.

Figure 3.

Examples of transcript isolation and thresholding in A1. A, Young tissue. B, Aged tissue. Boxes represent locations of panels centered on L4 (1) and CA2 hippocampus (2). A1′, A2′, B1′, B2′, Raw images from A and B. A1″, A2″, B1″, B2″, Images after color thresholding. A1″′, A2″′, B1″′, B2″′, Images after binary thresholding and watershed separation. Transcript counts derived from particle counts by cortical layer. For details, see Materials and Methods. Scale bars: A, B, 250 um; A1′–B2″′, 100 um.

Figure 4.

BF cholinergic inputs to A1. A, C, ChAT (magenta) and NeuN (blue) IF in A1. B, D, AChE histochemistry. C, D, Grayscale density profiles for ChAT and AChE across layers. E, AAV (pAAV-Ef1a-DIO-hChR2(H134R)-EYFP-WPRE-pA) injection in BF (arrow) of a ChAT-Cre Long-Evans rat(LE-Tg(Chat-Cre)5.1Deis) with ChAT IF (green) in A1. F, Cholinergic inputs from BF (green) to an exemplar L5B pyramidal neuron (red) that was strongly depolarized by puffed ACh. Colocalization (yellow) of the BF inputs and L5B neuron was shown in both dendrites (F, top right) and soma (F, bottom right). Laminar boundaries indicated in transparent vertical bars. wm, White matter. Scale bars: A, B, 25 µm; C, D, 250 µm; E, 40 µm.

Figure 5.

Examples of cellular phenotyping in A1 neurons. A, Coexpression of β2 (yellow dots), α4 (red dots), and α7 (aqua dots) in L4 of A1. Arrows indicate cells containing β2 and α4, but not α7 transcripts. B, Coexpression of β2 with VGluT1 (red dots) and VGAT (aqua dots). β2 (yellow dots) is expressed in several glutamatergic (VGluT1⁺) neurons, and 3 VGAT⁺ neurons (arrows). Light gray represents DAPI. Scale bars, 10 µm.

Figure 6.

nAChR subunit coexpression in A1 cells. A, Merged plot of all cells containing β2, α4, or α7 transcripts. B, Plots of cells with subunit combinations shown in D–H. C, β2⁺α4⁺* cells (black circles). *Any other transcript combination. D, β2⁺α4⁺α7^– (blue) and β2⁺α4^–α7⁺ (green) cells. E, β2⁺α4⁺α7 cells (black). F, β2⁺α4⁺α7^– cells (blue). G, β2⁺α4^–α7⁺ cells (green). H, β2^–α4^–α7⁺ cells (red). I, Chart summarizing the proportion of total cells that expressed each subunit combination by A1 layer (mean of 4 animals + SD).

Figure 7.

nAChR subunit coexpression by cell class in A1. A–I, nAChR transcript expression plotted by cell class (VGluT1 or VGAT), where each symbol represents one cell. Left column, Cells of any type containing β2, α4, or α7 transcripts. Middle column, VGluT1+ cells that did or did not coexpress β2, α4, or α7 transcripts. Right column, VGAT+ cells that did or did not coexpress β2, α4, or α7 transcripts. J–O, Charts summarizing the proportions of cells that expressed each subunit, by cell class and layer. *SDs not calculated due to insufficient numbers of L1 VGluT1+ cells. Scale, 1 mm.

Figure 8.

β2, α4, or α7 nAChR transcript density using ISH is depleted across A1 layers in aged animals. Normalized mean transcript density (mean ± SD) for β2 (left), α4 (middle), and α7 (right) in young (dark blue) and aged (light blue) A1 layers demonstrate significant downregulation of nAChR subunits with aging. *p < 0.05.

Figure 9.

Significant loss of binding sites and increase in affinity with aging. A, Exemplar autoradiograph (375 pm [³H]epibatidine) of a cross-section through A1 shows high levels of nAChRs in the deeper A1 layers from a young FBN rat. Significant nAChR binding is also seen in the MGB, superficial layer of the superior colliculus (SC), and the interpeduncular nucleus (IPN). Nonspecific binding was measured in the presence of 300 μm nicotine. B–E, Saturation binding curves show significant age-related loss of nAChRs density (B_max) across A1 layers plotted in histogram F. There was a significant age-related increase in affinity (decrease in K_d) in L5 with no significant changes seen in L4 and L6 (G). *p < 0.05.

Figure 10.

Age-related loss of differential nAChR mediates PSCs in L5 pyramidal neurons. Exemplar traces represent postsynaptic responses to local ACh application at 0.01 (pink), 0.05 (blue), 0.1 (green), 0.5 (purple), and 1.0 (red) mM from one young (A, top traces) and one aged (A, bottom traces) L5 pyramidal neuron, voltage-clamped at −70 mV. The mAChR blocker, atropine (Atr, 20 μm), was present in all experiments. B, Dose–response curves compare local ACh application onto young (red, n = 15, 7 rats) and aged (blue, n = 15, 7 rats) L5 pyramidal neurons. A1 neurons showed a significant age-related (Two-way ANOVA, F_(1,133) = 9.44, p = 0.003, Two-way ANOVA, dose, age group) and single-point difference in peak amplitude at 1 μM (One-way ANOVA, dose 1.0, F_(1,28) = 6.89, p = 0.014). C, Mean peak amplitude from L5 pyramid neurons in the presence of selective β2 (DHβE) and α7 (MLA) blockers in young and aged animals. Puffed ACh induced significantly smaller peak amplitude in neurons from aged rats than in neurons from the young rats (t₍₄₀₎ = 3.62, p = 0.001, t test). DHβE dramatically inhibited the PSCs in neurons from young rat (t₍₃₀₎ = 6.09, p < 0.0001, t test), with less of an effect in neurons from aged rats (t₍₂₈₎ = 2.3, p = 0.025, t test). While MLA significantly inhibited PSCs in pyramidal neurons from aged animals (t₍₂₈₎ = 3.44, p = 0.002, t test), a much smaller percentage blockade was seen in the reduction of PSCs from young animals (t₍₃₂₎ = 2.07, p = 0.046, t test). D, The percent change by selective nAChR blockade showed a significant age-related increase in relative MLA inhibition of the PSCs (t₍₂₀₎ = 2.14, p = 0.04, t test). All the recordings were made in the presence of atropine. *p < 0.05, **p < 0.01, ***p < 0.001, ns = nonsignificant.