VTA dopamine neurons are hyperexcitable in 3xTg-AD mice due to casein kinase 2-dependent SK channel dysfunction

Abstract

Alzheimer's disease (AD) patients exhibit neuropsychiatric symptoms that extend beyond classical cognitive deficits, suggesting involvement of subcortical areas. Here, we investigated the role of midbrain dopamine (DA) neurons in AD using the amyloid + tau-driven 3xTg-AD mouse model. We found deficits in reward-based operant learning in AD mice, suggesting possible VTA DA neuron dysregulation. Physiological assessment revealed hyperexcitability and disrupted firing in DA neurons caused by reduced activity of small-conductance calcium-activated potassium (SK) channels. RNA sequencing from contents of single patch-clamped DA neurons (Patch-seq) identified up-regulation of the SK channel modulator casein kinase 2 (CK2). Pharmacological inhibition of CK2 restored SK channel activity and normal firing patterns in 3xTg-AD mice. These findings shed light on a complex interplay between neuropsychiatric symptoms and subcortical circuits in AD, paving the way for novel treatment strategies.

Figure 1.. Instrumental reward learning is age-dependently impaired in 3xTg mice.

a. Schematic of procedure used for operant self-administration of palatable food pellets. b. 3xTg mice acquired operant self-administration of palatable pellets more slowly than WT mice (main effect, P = 0.0007, F_1,44 = 13.27), and older mice learned the task more slowly than younger mice (main effect, P = 0.0156, F_1,44 = 6.34). c. Raster plots showing correct side nose poke responding (vertical ticks along the x-axis) on day 1 of training for individual mice (by row) that eventually acquired self-administration. While there is considerable variation within the 3xTg groups, overall, they show less responding on day 1 compared to WT mice. d. Although there was a significant effect on learning the operant task, the number of pellets earned at FR30 did not significantly differ by age (P = 0.337) or genotype (P = 0.600). e. The breakpoint during the PR test session was not different by age (P = 0.402) or genotype (P = 0.483).

Figure 2.. VTA DA neurons from 3xTg mice display increased firing rates and decreased regularity.

a. Schematic of a cell-a Sached recording and 10 s representative traces of spontaneous firing activity from 12 mo WT (black) and 3xTg (red) mice. b. Mean spontaneous firing rate for 3, 6, 12, and 18 mo WT and 3xTg DA neurons (N = 576 cells, two-way ANOVA F_genotype = 9.236, P=0.0025; F_age = 3.878, P=0.0092; F_interac(on = 5.943, P=0.0005), with the most notable increase in rate at 12 mo (P<0.0001). c. Mean coefficient of variation of the ISI (CV_ISI) collected for the same cells as in b (F_genotype = 19.64, P <0.0001), and showed significant difference between 3xTg and WT at 12 mo (P=0.0214). d. Schematic of a whole-cell recording and representative traces of +100 pA current injection for 1 s in WT (black) and 3xTg (red) mice. e. Averaged frequency/current (F/I) curves from 12 mo WT and 3xTg mice (n=24 WT and n=31 3xTg, F_genotype = 54.56; P<0.0001, F_{current injection} = 18.48, P<0.0001). f. Hypersensitivity of 3xTg DA neurons measured as the slope of the F/I curve (0-60 pA, N = 222 cells, F_genotype = 13.71, P=0.0003; F_age = 2.692, P=0.0471), with a significant increase in sensitivity at 18 mo in 3xTg DA neurons (P=0.0044). g. Schematic of perforated-patch gramicidin recording and representative traces from spontaneously firing DA neurons from WT (black) and 3xTg (red) mice. h. Perforated-patch recordings show increased spontaneous firing frequency in 3xTg neurons (n = 38 WT and 35 3xTg neurons, two-tailed t-test, P<0.0001), and i. increased CV_ISI (two-tailed t-test, P=0.0363). j. Depolarized apparent threshold in 3xTg DA neurons (two-tailed t-test, P=0.0247). k. Grand averages of ISI voltage trajectories for WT and 3xTg cells. l. Depolarized mean medium aeer-hyperpolarization voltage (mAHP, two-tailed t-test, P<0.0001), and m. minimum ISI voltage (two-tailed t-test, P<0.0001) in 3xTg DA neurons.

Figure 3.. SK channel inhibiGon in WT neurons recapitulates the 3xTg phenotype.

a. Representative traces of the tail current in response to a 100 ms depolarization step to −17 mV from V_hold (−72 mV) in the whole-cell configuration. b. Reduced tail current maximal amplitude (N = 251 cells, two-way F_genotype = 8.743, P=0.0034; F_age = 10.57, P<0.0001) and c. area under the curve (AUC, F_genotype = 13.79, P=0.0002, F_age = 10.03, P<0.0001) in 3xTg DA neurons. d. Perforated-patch recordings from spontaneously firing WT cells in control ACSF (black) and in the presence of apamin (1-3 nM) (orange). e. Increased firing frequency (paired two-tailed t-test, P=0.0067) and f. C_VISI (paired two-tailed t-test, P=0.0088) in presence of apamin. g. Depolarized apparent spike threshold in response to apamin (paired two-tailed t-test, P=0.0050). h. ISI voltage trajectory averages. The effect of apamin is reminiscent of the trajectories from 3xTg neurons (panel 1k). i. Depolarized mAHP (paired two-tailed t-test, P=0.0022) and absolute minimum ISI voltages (paired two-tailed t-test, P=0.0018) in apamin.

Figure 4.. NS309 efficacy is decreased in 3xTg DA neurons.

a. Representative traces of WT DA neuron pacemaking in control ACSF (black) and in the presence of NS309 (1 μM) (blue). b. Representative traces of 3xTg DA neurons in control ACSF (red) and in NS309 (gray). c. Firing frequency was decreased by NS309 (two-way RM ANOVA, FNS309 = 15.27, P = 0.0058; F_genotype = 5.872, P=0.0459; F_{NS309 x genotype} = 6.604, P=0.0370) in WT (P=0.0007) but not significantly in 3xTg (P=0.0554) neurons. d. NS309 did not affect CV_ISI. e. Averaged ISI voltage trajectories in WT neurons before (black) and aeer NS309 (blue) and f. in the 3xTg group before (red) and in the presence of NS309 (gray). g. NS309 hyperpolarizes mean mAHP voltages in WT (P<0.0001), but not 3xTg (P=0.7795) neurons (tw-oway RM ANOVA, F_NS309 = 9.963, P=0.0160, F_genotype = 14.89, P=0.0062, F_{NS309 x genotype} = 43.200, P=0.0003). h. NS309 hyperpolarizes minimum ISI voltages (F_genotype = 11.06, P=0.0127; F_{NS309 x genotype} = 11.94, P=0.0106) in WT (P=0.0037) but not 3xTg (P=0.9988) neurons.

Figure 5 –. Patch-seq analysis indicates up-regulated Csnk2a1 transcripGon.

a. For Patch-seq, recording from an individual neuron is followed by cell extraction with the recording pipeSe. Cell contents are captured, and each individual cell RNA-Seq library is prepared and sequenced. b. Individual cells projected in transcriptome PCA space and sized by Tail Current AUC (pA s) demonstrated some separation of 3xTg and WT. c. Volcano plot of differentially expressed genes (t-test, B-H MTC, FDR<0.1, ∣FC∣>1.25). d. Venn diagram describing overlap in differentially expressed genes between groups and those correlating to Tail Current AUC. e. Volcano plot describing molecular function gene ontology analysis (weighted set cover redundancy reduction) on DEGs indicating increased ion channel binding (GO:0044325) pathways (FDR=0.0089544, Normalized Enrichment Score=2.1932). f. Example differentially expressed genes (Csnk2a1, Grk4, and Cacna2d1). *FDR<0.1, **FDR<0.05.

Figure 6.. Casein kinase 2 inhibiGon restores firing properGes in 3xTg DA neurons.

a. Representative firing trace from a naive WT neuron (black) and one pre-incubated with SGC (1 μM, purple). b. Representative trace from a naive 3xTg neuron (red) and one pre-incubated with SGC (gray). c. SGC decreased firing frequency in 3xTg (P<0.0001) but not WT (P=0.1140) neurons (N = 103 cells; F_genotype = 5.988, P=0.0161; FSGC = 3.514, P=0.0637; F_{genotype x SGC} = 18.93, P<0.0001). d. SGC decreased CV_ISI in 3xTg (P=0.0002) but not WT (P=0.9796) DA neurons (N = 103 cells, FSGC = 9.502, P=0.0026; F_{genotype x SGC} = 8.034, P=0.0055). e. Averaged ISI voltage trajectories of naïve 3xTg neurons and ones pre-incubated with SGC. f. SGC hyperpolarized the mAHP in 3xTg (P<0.0001) but not WT (P=0.3225) neurons (F_genotype = 6.349, P=0.0133; FSGC = 36.74, P<0.0001; F_{genotype x SGC} = 17.22, P<0.0001). g. Pre-incubation with SGC restored tail current charge in 3xTg (P=0.0098) neurons but had no effect in WT (P=0.9257) neurons (N=84 cells, F_{genotype x SGC} = 4.765, P=0.0318). h. Principal component analysis of electrophysiological properties in all groups. The first component describes 46.6% of the variability, and the second describes 23.1% of the variability. 3xTg and WT + apamin cells largely fall to the right, while WT neurons and manipulations as well as 3xTg + SGC segregate to the lee.

Figure 7.. Proposed mechanism of hyperexcitability in 3xTg VTA dopamine neurons.

Wildtype mice maintain a balance between CK2 and PP2A resulting in low basal phosphorylation of CaM in DA neurons. In 3xTg mice, hyperactive CK2 results in hyperphosphorylated CaM, effectively decreasing SK channel calcium binding affinity and channel opening. This results in increased firing rate and increased irregularity in 3xTg mice.