Dopamine Deficiency Reduces Striatal Cholinergic Interneuron Function in Models of Parkinson's Disease

Abstract

Motor and cognitive functions depend on the coordinated interactions between dopamine (DA) and acetylcholine (ACh) at striatal synapses. Increased ACh availability was assumed to accompany DA deficiency based on the outcome of pharmacological treatments and measurements in animals that were critically depleted of DA. Using Slc6a3^DTR/+ diphtheria-toxin-sensitive mice, we demonstrate that a progressive and L-dopa-responsive DA deficiency reduces ACh availability and the transcription of hyperpolarization-activated cation (HCN) channels that encode the spike timing of ACh-releasing tonically active striatal interneurons (ChIs). Although the production and release of ACh and DA are reduced, the preponderance of ACh over DA contributes to the motor deficit. The increase in striatal ACh relative to DA is heightened via D1-type DA receptors that activate ChIs in response to DA release from residual axons. These results suggest that stabilizing the expression of HCN channels may improve ACh-DA reciprocity and motor function in Parkinson's disease (PD). VIDEO ABSTRACT.

Keywords: HCN channel; RiboTag; acetylcholine; diphtheria toxin; dopamine acetylcholine ratio; dopamine depletion; electrophysiology; receptor.

Figure 1. Progressive DA deficiency in Slc6a3^DTR/+ mice.

A) Treatment timeline. B) Weights of treated mice remain stable over time. For all panels, n=mice. For all figures, data are represented as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, reserpine vs. WT_saline; ^!p<0.05, ^!!p<0.01, ^!!!p<0.001, DAT_dT vs. WT_dT; ^@p<0.05, ^@@p<0.01, DAT_dT-2w vs. DAT_dT-4w; ^$p<0.05, ^$$p<0.01, ^$$$p<0.001, DAT_dT-2w vs. reserpine. C) WT_saline and WT_dT mice show a progressive increase in the latency to fall from the rotarod over 3 consecutive trials. DAT_dT mice show a progressive reduction in rotarod performance. D) Rotarod performance is similar across control groups. Reserpine mice are incapacitated. E) Striatal DA content is depleted 12 hr after reserpine and progressively decreases 2 and F) 4 wk following the first injection of dT. G) DAT_dT-2w mice show impaired balance beam performance. H₁) Open field tests show an increase in ambulations, H₂) rearings, and H₃) stereotypies in DAT_dT-2w mice following 5 daily treatments with L-Dopa. See also Figure S1 and Table S1.

Figure 2. DA deficiency reduces ChI pacemaking.

A) Cell-attached recording (left) and a fluorescent ChI (right). Bar, 10 μm. B) Representative traces of whole-cell recordings from ChIs. C) The firing frequency is lower in ChIs from Slc6a3^DTR/+ mice examined 2 and 4 wk after treatment with dT. Box-and-whisker plots: boundary, 25th and 75th percentiles; median, solid black line; mean, solid red; whiskers, 10th and 90th percentiles; outlying points, red circles. n= cells. D) The plot shows a higher CV in ChIs from DAT_dT mice. E) Plots show a negative correlation between the CV and the firing rate of ChIs from control, F) reserpine, and G) DAT_dT mice. Lines, linear regression. See also Figure S2 and Table S2.

Figure 3. HCN channels regulate ChI firing.

A) Simplified ChI with DA receptors and HCN channel. B) The panel illustrates Na⁺, K⁺, Ca²⁺, and Ihcn-dependent firing in ChIs. The Na⁺ channel generates the spike, which activates Ca²⁺ channels that shape the AP. A- and BK-type K⁺ channels promote recovery and hyperpolarization. Spike-driven Ca²⁺ activates SK channels, producing the after-hyperpolarization potential (AHP). Activation of the HCN channel then carries Na⁺, K⁺, and Ca²⁺ into the cell to produce a depolarizing ramped inward current (Ihcn). C) Traces of a perforated-patch recording in a ChI shows the inward Na⁺ channel current (upper) in response to a depolarizing current injection (lower). TTX eliminates the inward current (middle). D) Spontaneous firing in a ChI (upper) is abolished by TTX (lower), with no observable washout. E) Whole-cell recordings in ChIs show that depolarizing voltage injections create an inward current that is blocked by TTX. For all panels, n=cells. F) Subtraction of TTX from baseline reveals the Na⁺ channel current, which increases in response to greater depolarizing voltage. G) The slow relaxation of the membrane current (upper) in response to a hyperpolarizing voltage (lower) is blocked by Cs⁺ (middle). H) ChI firing (upper) is reversibly inhibited by Cs⁺. I) Whole-cell voltage clamp recordings show that Cs⁺ reduces currents in response to ChI hyperpolarization. J) Subtraction of Cs⁺ from baseline reveals Ihcn that increases in response to greater hyperpolarizing voltages.

Figure 4. DA deficiency reduces Ihcn.

A) Voltage clamp recordings in ChIs demonstrate B) similar eRMPs across the groups of mice. For all panels, n=cells. C) Current clamp recordings in ChIs D) demonstrate similar Rin across the groups of mice. E₁) Representative voltage clamp recording in a ChI from a control mouse demonstrates the slow relaxation of membrane current (Ihcn; top trace) in response to hyperpolarizing voltage (bottom). Ihcn was measured 350 ms after the change in voltage (black arrow). Ihcn flow (pA/s) was measured over 900 ms (red arrow heads). E₂) Representative trace of a ChI from a reserpine mouse shows a reduced Ihcn flow and a lower number of spikes in response to depolarizing voltage, suggesting a reduction in Ihcn kinetics and excitability. E₃) Ihcn flow and spiking is further reduced in a ChI from a DAT_dT mouse. F) Ihcn and G) Ihcn kinetics (Ihcn/Imax) are reduced in ChIs from reserpine and DAT_dT mice. H) Ihcn flow is reduced in ChIs from reserpine mice and progressively diminishes in DAT_dT mice. I) The HCN channel blockers Cs⁺ or ZD7288 eliminate Ihcn flow. J) Subtraction of any residual currents following Cs⁺ or ZD7288 has no effect. K) Isolation of the TTX-sensitive current by subtraction shows that DA deficiency does not affect Na⁺ channel function. L) The plot shows the zero-crossing point between Cs⁺- and Na⁺-sensitive inward currents in ChIs from control mice. The intercept between these two excitatory currents lies at a progressively positive voltage in ChIs from M) reserpine and N) DAT_dT mice, leading to a lower spike frequency. See also Table S3.

Figure 5. DA deficiency reduces Chat, Ache, and Hcn subunit expression.

A) HCN and Na⁺ channel modulation by D1 and D2 receptors. cAMP binds to the HCN CNBD to reduce inhibition of HCN channel gaiting. AC, adenylyl cyclase. B) HA.11 antibody-tagged ChIs from a RiboTag × ChAT-Cre × tdTomato mouse. Bar, 20 μm. C) A single, sharp peak in the melting curve analysis following the RT-qPCR reaction shows that only one product is generated from each primer pair. D) RT-qPCR amplification curves of Chat, Hcn1–4, and Musr18S expressed in ChIs. The number of cycles required to cross C_T is inversely proportional to RNA expression. E_1–4) Relative mRNA expression (2^−ΔC_T) of the Hcn1–4 subunits, where ΔC_T is determined by subtracting C_T from the Musr18S control. DA-deficient mice were processed with their respective controls. For all panels, n=mice. F) Hcn1–4 expression in striata from WT_saline and WT_dT controls, reserpine, and DAT_dT mice. ^&p=0.04, 2-way ANOVA. G₁) Fold-reduction (2^−ΔΔC_T) of mRNA in ChIs from reserpine, G₂) DAT_dT-2w, and G₃) DAT_dT-4w mice relative to their controls. H₁) Charts illustrate changes in the percent distribution of Hcn1–4 subunits based on their gene expression in control, H₂) reserpine, H₃) DAT_dT-2w, and H₄) DAT_dT-4w mice. I) mRNA expression of Chat in ChIs from control mice, and J) Chat, K) Ache, and L) Vachat relative to their controls. See also Table S4.

Figure 6. Motor performance is dependent on DA and ACh availability.

A) The latency to fall from an accelerating rotarod increases over three consecutive trials. Motor learning is poor in DAT_dT-2w mice. For all panels, n=mice. B) The average latency to fall from the rotarod is reduced in DAT_dT-2w mice. C) Striatal DA content is depleted 12 hr following reserpine and is moderately reduced in DAT_dT-2w mice. D) ACh content is reduced in reserpine and DAT_dT-2w mice. E) The ACh/DA ratio is increased in DAT_dT-2w mice and rises further in reserpine mice. F) Latency to fall in control mice shows no correlation with striatal DA content, G) ACh content, or H) the ACh/DA ratio. Lines in panels F-K, linear regression. I) For DAT_dT-2w mice, the latency to fall positively correlates with striatal DA content and J) negatively correlates with ACh content and K) the ACh/DA ratio. L) The plot compares the latency to fall with DA content, M) ACh content, and N) the ACh/DA ratio. Bars, SE; ^&&p<0.01, 2-way rm-ANOVA.

Figure 7. DA deficiency modifies DA receptor responses.

A) Representative cell-attached recordings of ChIs and graph show slower firing in ChIs from DAT_dT-2w mice. B) Amph reduces ChI firing in WT_dT-2w mice and C) increases firing in DAT_dT-2w mice. For all panels, n=cells; ^#p<0.05, ^##p<0.01, ^###p<0.001, vehicle compared to ligand, paired t-test. D) Percent change of ChI firing in response to Amph from WT_dT-2w and DAT_dT-2w mice. E) Correlation between the baseline firing rate and the percent change in firing following Amph in ChIs from WT_dT-2w and F) DAT_dT-2w mice. Lines, linear regression. G) Firing in ChIs from WT_saline, H) reserpine, and I) DAT_dT-2w mice increases in response to the D1R agonist (SKF) and decreases in response to the D2R agonist (Quin). J and K) SKF boosts firing in ChIs from DAT_dT-2w mice. ^&F_(2,18)= 3.66, p=0.04, 2-way ANOVA. L and M) Quin reduces firing in ChIs from reserpine mice. ^&F_(2,24)= 6.89, p=0.02, 2-way ANOVA. See also Figure S4.

Figure 8. DA receptors modulate ChIs.

A) The I-V curves show current induced by SKF81297 crosses the 0 pA threshold earlier in DAT_dT-2w mice and cAMPS has no effect. For all panels, n=cells. B) The plot shows the zero-crossing point between Na⁺ channel- and SKF81297-dependent inward currents in ChIs from control mice. The intercept between these two excitatory currents lies at a progressively negative voltage in ChIs from C) reserpine and D) DAT_dT-2w mice. E) Quinpirole produces a net inhibitory outward current at potentials close to depolarization. cAMPS blocks this outward current and unmasks a net inhibitory current at hyperpolarized potentials. F) ChI firing in response to applied input current (left) is reduced in ChIs from DAT_dT-2w mice (right). G) The illustration shows that acute-severe DA depletion reduces Chat (dots) and Ihcn to decrease ACh output. DA released from residual boutons weakens ACh release via D2Rs. Moderate-progressive DA deficiency further reduces Ihcn and ACh output; evoked DA release increases ACh efflux via D1Rs. Severe-progressive DA deficiency reduces Chat and Ache and further diminishes Ihcn. See also Figures S5–S8 and Tables S5–S7.