Animal care was conducted in accordance with the United States Public Health Service Guide for the Care and Use of Laboratory Animals, and procedures were pre-approved by the Institutional Animal Care and Use Committee at the University of California Los Angeles. Transgenic mice overexpressing human alpha-synuclein under the Thy1 promoter (Thy1-aSyn) were crossed into a hybrid C57BL/6-DBA/2 background (16). Animals were maintained on the hybrid C57BL/6-DBA/2 background by breeding mutant females with male mice on the hybrid background (18, 24, 25). Littermates were never inbred. The genotype of all Thy1-aSyn and WT mice was confirmed with polymerase chain reaction (PCR) amplification analysis of DNA from tail tips at birth and confirmed at the end of the experiment. All mice were individually housed with free access to water and standard rodent chow.

Male mice from a total of 21 litters were used in this study. Litter sizes ranged from 2 to 11 mice. Separate cohorts of mice were used in the hemodynamic-baroreflex protocol (Thy1-aSyn = 9, WT = 9) and for the heart rate (HR) and contractility dobutamine challenge protocol (Thy1-aSyn = 7; WT = 7) at 9–12 months of age. An additional separate cohort of mice (Thy1-aSyn = 3–4, WT = 3–5) was implanted with arterial transducers and tested at 3–5 months of age. A younger cohort in the arterial transducer experiment was included because the survival rate in older animals was too low.

Following hemodynamic assessment surgeries Thy1-aSyn and WT mice were euthanized and heart, lung, and liver weights were measured and tibia length determined.

Hemodynamic assessment was performed as previously described (26–29). Briefly, in Thy1-aSyn and WT mice anesthetized with ketamine, xylazine, and buprenorphine, both femoral arteries were catheterized with flame-stretched PE-50 tubing (30). One catheter was interfaced to a pressure transducer connected to the PC computer data acquisition system. The other catheter was used for drug injections. The functionality of the sympathetic and parasympathetic branches of the autonomic nervous system were independently evaluated by intravascular injections of the vasoconstricting α-1 adrenergic receptor agonist phenylephrine (5 and 25 μg) and the vasodilating nitric oxide donor sodium nitroprusside (5 and 15 μg). This was followed by autonomic nervous system blockers glycopyrrolate (a peripheral acting quaternary amine muscarinic receptor antagonist, 20 μg) and propranolol (β-adrenergic receptor antagonist, 50 μg) and reinfusions of phenylephrine and sodium nitroprusside to confirm abolition of the baroreceptor reflex. All drug injections were administered intrarterially in a volume of 100 μl of 0.9% aqueous saline heparinized at 7 U/ml and injected over 7 s. HR was determined from the beat-to-beat systolic pressure peak intervals.

We used dobutamine to probe the integrity of the cardiac β-1 adrenergic receptor as reflected in HR and rate of left ventricular pressure change during systole (inotropic response) and diastole (lusitropic response). Thy1-aSyn and WT mice were anesthetized with ketamine, xylazine, and buprenorphine and placed on a warming pad. HR and temperature were continuously monitored during surgery. Once anesthetized, the right carotid artery was catheterized and a 1.4 Fr catheter (Millar Instruments, Houston, TX, USA) was advanced into the left ventricle to obtain a dynamic pressure signal that was mathematically differentiated into in vivo indices of cardiac muscle inotropy (+dP/dT) and lusitropy (−dP/dT) (26). A femoral artery was also catheterized as above for injection of dobutamine, a β-1 adrenergic receptor agonist. Dobutamine was injected in serial doses of 3, 6, 15, and 30 ng/g with at least a 5-min recovery period between injections as an adrenergic post-synaptic receptor challenge test. Measurements were acquired with Hem Software (Notocord Systems V4.2, Croissy sur Seine, France). HR and left ventricular pressure were recorded 150 s after injections and averaged over a 10-s period. Maximum and minimum dP/dT were calculated during this 10-s period.

Arterial pressure and cage activity was measured simultaneously in awake, freely moving Thy1-aSyn and WT mice using radio telemetry transducers (TA11PA-C10; Data Sciences International Inc., St. Paul, MN, USA). Transmitter units were implanted subcutaneously along the mouse’s ventral side under the ketamine, xylazine, and buprenorphine anesthesia. The transmitter blood pressure catheter was fed over the shoulder and inserted into the left carotid artery under sterile surgical conditions (30). Data recording from the mice began immediately following implantation via an antenna receiver under the cage connected to a computer system. Pressure waveforms and activity data were collected, analyzed, and displayed with the telemetry software. For all studies, 20 s of data were collected every 10 min for the duration of the study. Data waveforms and parameters were analyzed with the DSI analysis program (ART 4.0) to determine differences in systolic, diastolic, and mean arterial pressures plus cage activity and HRs between Thy1-aSyn and WT mice during the entire light-dark diurnal cycle. Data for the hours surrounding the weekly scheduled cage changing were excluded from analysis. The data were averaged at the same time of day to create “foldagrams” to reveal any variances (dysfunction) and compare changes between groups under baseline conditions (30).

We tested tonic vagal release of acetylcholine at the muscarinic receptor of cardiac pacemaker cells with the muscarinic antagonist atropine [1 mg/kg, ip; (31)] administered to both Thy1-aSyn and WT mice. We documented blood pressure and baroreceptor reflex induced HR changes after phenylephrine [3 mg/kg, ip; (32)] and sodium nitroprusside [1 mg/kg, sc; (33)]. Each drug was administered at the same time of day at least 2 days apart to allow for complete drug clearance from the ip injection. Saline was used as the vehicle and administered before each drug injection. Blood pressure and HR were recorded continuously in the home cage, following saline, and following drug administration.

The hemodynamic experiment plots (Figures 1A–F) depict the baseline and acute post-drug pressure and HR data pairs as means and standard errors, computed as a function of condition, based on the nine mice in each experimental cell. The slope and R² statistics were derived with standard bivariate linear regression analyses, determined from the raw systolic and beats per minute data pairs, as a function of the genotype × drug conditions, but collapsed across the three doses of each drug (i.e., n = 27 for each genotype × drug cell). These and the chronic telemetric data are also expressed as the ratio of the change in HR in BPM to the change in systolic blood pressure in mmHg, or ΔHR/ΔSBP. For the dobutamine challenge test and cardiac contractility experiments data are expressed as left ventricular +dP/dT_max and −dP/dT_min. A mixed design ANOVA was used to compare genotypes and dose of drug in the hemodynamic and contractility experiments. Student’s t-test or Mann–Whitney U were applied to analyze morphometry parameters and arterial telemetry data between Thy1-aSyn and WT mice. Fisher’s Least Significant Difference was used for post hoc analyses. All statistics were calculated with GB-Stat software (Dynamic Microsystems, Inc., Silver Spring, MD, USA, 2000). The level of significance was set at p < 0.05.

Morphometry measures included measurements of body, heart, liver, and lung weights, and tibia length in Thy1-aSyn and WT mice (Table 1). Comparisons between WT and Thy1-aSyn mice using Student’s t-test showed that Thy1-aSyn mice weighed significantly less than WT mice, a difference we reported previously in this mutant line (19). While there were no significant differences in any of the other measures, heart and liver weights tended to be smaller in Thy1-aSyn mice but the difference did not reach significance (p = 0.08 and p = 0.06, respectively).

Baseline systolic blood pressure measurements did not differ between conscious or anesthetized WT and Thy1-aSyn mice (p > 0.05). However, baseline HR measures were significantly different between genotypes under anesthesia during the acute hemodynamic studies (Table 2); Thy1-aSyn mice had a higher HR compared to WT mice (p < 0.05). In contrast, conscious WT and Thy1-aSyn mice equipped with arterial telemetry had comparable baseline HRs during both the light and dark cycles (Table 2).

The baroreflex response to the injection of phenylephrine and sodium nitroprusside in anesthetized Thy1-aSyn and WT mice are plotted in Figures 1A,B. Though the plots show averaged pressure and heart values for each condition for clarity, the individual baseline systolic pressures were measured over a wide range of values from 48 to 159 mmHg in WT mice and from 41 to 157 mmHg in Thy1-aSyn mice. Linear regression analysis from the raw data pairs (n = 27 pairs per study) are drawn through the averaged data points under each condition in Figures 1A,B. These data are also plotted as the ΔHR/ΔSBP score (baroreflex sensitivity index) in Figures 1C,D. These data clearly show that phenylephrine produced a similar increase in systolic blood pressure and reflex decrease in HR in WT and Thy1-aSyn mice resulting in ΔHR/ΔSBP scores that did not differ between genotypes over this range of baseline pressures (Figure 1C). As expected with combined β-adrenergic and muscarinic receptor blockade, the ΔHR/ΔSBP response to phenylephrine following β-adrenergic and muscarinic blockade also did not differ between genotypes (Figure 1C). In contrast, the response to sodium nitroprusside in WT mice showed the characteristic decrease in systolic blood pressure and increased HR, while in Thy1-aSyn mice systolic blood pressure decreased but HR remained close to their initial baseline values regardless of dose (Figures 1B,D). Here, Thy1-aSyn regression slope was very shallow and ΔHR/ΔSBP scores were significantly lower compared to WT mice (p < 0.05). The ΔHR/ΔSBP response to sodium nitroprusside following β-adrenergic and muscarinic blockade did not differ between genotypes (Figure 1D).

Analysis of HR in WT and Thy1-aSyn mice showed phenylephrine caused similar HR decreases in both genotypes following administration of phenylephrine [Figure 1E; F(1,16) = 4.33, p > 0.05 for genotype and F(2,32) = 39.47, p < 0.01 for drug dose]. However, analysis of HR following sodium nitroprusside revealed that WT but not Thy1-aSyn showed significant increases in HR after each dose [Figure 1F; F(1,16) = 1.69, p > 0.05 for genotype and F(2,32) = 25.65, p < 0.01 for drug dose, and F(2,32) = 7.34, p < 0.05 for genotype × drug dose interaction].

Left ventricular inotropy (contraction) and lusitropy (relaxation) (±dP/dT) were measured in WT and Thy1-aSyn mice at 9–12 months of age (Figure 2). Serial infusions of the β-adrenergic agonist dobutamine resulted in similar left ventricular contractility (+dP/dT; Figure 2A) and relaxation (−dP/dT; Figure 2B) between WT and Thy1-aSyn mice (p > 0.05). HR responses were also similar between genotypes (p > 0.05; Figure 2C).

Because Thy1-aSyn mice had a higher HR at baseline under anesthesia we wanted to determine if the differences we observed were due to an abnormal response to the anesthesia or to impaired baroreflex. Therefore, a separate cohort of Thy1-aSyn and WT mice were implanted with arterial telemetry transducers. Due to poor post surgery survival at the 9- to 12-month age in Thy1-aSyn mice the separate cohort of mice for telemetry was younger in age, 3–5 months. Similar to the hemodynamic experiment under anesthesia, conscious mice were challenged pharmacologically with intraperitoneal phenylephrine and nitroprusside (ip). In addition the response to muscarinic blockade was measured using atropine. At 3–5 months of age baseline HR and systolic blood pressure did not differ between WT and Thy1-aSyn mice. Similar to the anesthetized experiment WT and Thy1-aSyn mice displayed comparable responses to phenylephrine and nitroprusside over a similar range of systolic pressures. There was no difference between the genotypes in ΔHR/ΔSBP or HR scores following phenylephrine injections (p > 0.05; Figures 3A,D). However, in response to sodium nitroprusside Thy1-aSyn mice once again showed a blunted HR response while WT showed characteristic tachycardia. Thy1-aSyn ΔHR/ΔSBP scores were significantly decreased compared to WT (p < 0.05; Figure 3B). Analysis of HR alone showed that HR significantly increased in WT but not in Thy1-aSyn mice following nitroprusside (p < 0.05; Figure 3E). In response to muscarinic blockade ΔHR/ΔSBP scores did not significantly differ between genotypes however, analysis of HR alone showed that HR significantly increased in WT but not in Thy1-aSyn mice following atropine (Figures 3C,F). The activity response did not significantly differ between genotypes in any of the drug conditions (Student’s t-test, p > 0.05).