Main Research Findings

1) Administration of trihexyphenidyl HCl was shown to improve deficits in dopamine neurotransmission in models of dystonia.

2) Trihexyphenidyl HCl was found to improve symptoms of Parkinson’s disorder by reducing regional cerebral blood flow and regional cerebral metabolic rate, without additional cognitive impairments.

Selected Data
1) The study completed by the research team of Downs et al investigated how trihexyphenidyl (THP) affects dopamine (DA) neurotransmission in a mouse model of DYT1 dystonia, using a combination of ex vivo and in vivo neurochemical techniques. The experimental model consisted of Dyt1 knock-in mice heterozygous for the ΔE torsinA mutation (Tor1a+/ΔE), which mimics the human genetic condition, along with wild-type littermate controls (Tor1a+/+). All mice were bred on a C57BL/6J background and maintained under standard laboratory conditions, including a 12-hour light/dark cycle with free access to food and water. Both male and female mice aged 12–14 weeks were used. Genotyping was performed using PCR to confirm the presence or absence of the mutation. All procedures were approved by the institutional animal care committee [1].
To assess dopamine release, the researchers employed fast scan cyclic voltammetry (FSCV) in acute brain slices. Mice were euthanized and their brains rapidly removed and sectioned into 300 μm slices in ice-cold, oxygenated sucrose-based artificial cerebrospinal fluid (aCSF). Slices containing either the dorsolateral striatum or the nucleus accumbens (NAcc) were identified using anatomical landmarks. After recovery in oxygenated aCSF, slices were transferred to a recording chamber maintained at 32°C. A carbon fiber electrode was inserted into the tissue to detect dopamine, while a stimulating electrode was positioned nearby to evoke release. Electrical stimulation protocols included single-pulse or five-pulse bursts at 100 Hz, applied at intervals to prevent signal degradation. Dopamine oxidation and reduction currents were measured with high temporal resolution, and electrodes were calibrated using known dopamine standards [1].
Pharmacological manipulations were performed by bath-applying drugs directly to the slices. THP was tested across a range of concentrations between 3–300 nM to generate dose-response curves. Additional compounds included atropine, another muscarinic antagonist, nicotine, L-DOPA, administered systemically prior to slice preparation, and various receptor antagonists such as DHβE, a nicotinic receptor blocker, and CNQX, a glutamate receptor antagonist) Drugs were allowed to equilibrate before recordings began.
To complement ex vivo findings, in vivo microdialysis was conducted to measure extracellular dopamine levels in awake, behaving mice. A microdialysis probe was surgically implanted into the dorsal striatum under anesthesia. After recovery and overnight habituation, baseline samples were collected at 20-minute intervals. THP was administered either systemically (intraperitoneal injection) or locally via reverse dialysis through the probe. Dialysate samples were collected over a two-hour period and stored for analysis.
Dopamine levels in microdialysis samples were quantified using high-performance liquid chromatography (HPLC) with electrochemical detection. The system included a specialized column and detector setup capable of distinguishing monoamines based on retention time and electrochemical properties. The mobile phase composition and flow rate were carefully controlled to ensure accurate separation and detection. To investigate cholinergic system function, the researchers measured acetylcholinesterase (AChE) activity in striatal tissue using a colorimetric assay. Tissue homogenates were incubated with a substrate that produces a measurable color change upon enzymatic activity, allowing quantification relative to protein content [1].
Gene expression analysis was performed using quantitative real-time PCR (qRT-PCR) to assess mRNA levels of nicotinic acetylcholine receptor (nAChR) subunits (α4, α6, α7, and β2) in midbrain tissue. RNA was extracted, converted to cDNA, and amplified using specific primers. Expression levels were normalized to β-actin using the comparative CT method.
Statistical analyses included Student’s t-tests for pairwise comparisons and two-way ANOVA for experiments involving multiple variables, followed by post hoc tests where appropriate. Dose-response curves were analyzed using nonlinear regression to determine EC50 or IC50 values. Overall, the study combined electrophysiological, pharmacological, biochemical, and molecular approaches to comprehensively evaluate how THP influences dopamine release and how cholinergic mechanisms contribute to this effect in a dystonia model [1].
2) The study completed by researchers Takahashi et al was designed to investigate the effects of trihexyphenidyl, an anticholinergic medication, on regional cerebral blood flow (rCBF) and oxygen metabolism (rCMRO₂) in patients with Parkinson’s disease (PD) using positron emission tomography (PET). To ensure that the observed effects could be attributed specifically to the drug, the researchers recruited six previously untreated PD patients, consisting of four men and two women between the ages of 47 and 60 years, with a mean age of approximately 53.7 years. The duration of illness ranged from 10 to 48 months, and all patients displayed the hallmark motor features of Parkinson’s disease, including resting tremor, cogwheel rigidity, and akinesia. Although symptoms were present bilaterally, each patient exhibited asymmetry, with one side more severely affected [2].
Disease severity was assessed using the Hoehn and Yahr classification, with four patients categorized as stage II and two as stage III, indicating mild to moderate progression. Importantly, patients with confounding conditions such as drug-induced parkinsonism, vascular parkinsonism, multisystem disease, dementia with Lewy bodies, or clinical depression were excluded. Dementia was ruled out using DSM-III-R criteria, and depression was evaluated with the Hamilton rating scale. A control group of six neurologically healthy individuals, matched in sex distribution and similar in age range of 48 to 70 years, and a mean of 59.4 years, was also included for comparison. All participants provided informed consent prior to participation.
The experimental design employed a within-subject longitudinal approach, in which each patient was assessed before and after pharmacological treatment. Trihexyphenidyl was administered orally at a fixed dose of 6 mg per day for a period ranging from 5 to 11 weeks, with an average treatment duration of approximately seven weeks. Clinical disability, cognitive function, and PET imaging were evaluated both before the initiation of treatment and after the treatment period, allowing for direct comparison of pre- and post-treatment changes within the same individuals. Motor impairment was quantified using the Unified Parkinson’s Disease Rating Scale (UPDRS) alongside the Hoehn and Yahr staging system. Cognitive performance was assessed using a comprehensive neuropsychological battery that included the Mini-Mental State Examination (MMSE) for global cognitive screening, the Wechsler Adult Intelligence Scale–Revised (WAIS-R) for measures of intellectual functioning including verbal, performance, and full-scale IQ, and the Wechsler Memory Scale–Revised (WMS-R) to evaluate various aspects of memory, such as verbal and visual memory, delayed recall, and attention/concentration [2].
The neuroimaging component of the study relied on PET scanning to quantitatively measure cerebral hemodynamics and metabolism. Specifically, the researchers measured rCBF, rCMRO₂, regional oxygen extraction fraction (rOEF), and regional cerebral blood volume (rCBV) using a steady-state technique. During scanning, participants were placed in a quiet, darkened room and instructed to lie still with their eyes closed and covered, minimizing sensory stimulation and ensuring consistency across scans. Radiotracers were administered to obtain these measurements: carbon dioxide labeled with oxygen-15 (C¹⁵O₂) was used to assess cerebral blood flow, oxygen-15 gas (¹⁵O₂) was used to evaluate oxygen metabolism, and carbon monoxide labeled with oxygen-15 (C¹⁵O) was used to determine blood volume. The calculation of rCBV incorporated measurements of tracer concentration in both brain tissue and arterial blood, along with a correction factor for hematocrit differences between cerebral capillaries and larger blood vessels. Additionally, rOEF and rCMRO₂ values were corrected based on rCBV to improve accuracy.
PET images were acquired in 14 horizontal slices parallel to the orbitomeatal line, each with a thickness of 11 mm and spaced at intervals of 6.5 mm, covering a large portion of the brain from the base to the cortex. To analyze regional brain activity, the researchers defined multiple regions of interest (ROIs) across various anatomical areas, including the frontal, parietal, temporal, and occipital cortices, as well as subcortical structures such as the putamen, caudate nucleus, and thalamus, in addition to the cerebral white matter and cerebellum. Circular ROIs with diameters ranging from 12 to 18 mm were placed bilaterally in these regions. In patients with PD, hemispheres were categorized as contralateral or ipsilateral relative to the side of the body with the most severe motor symptoms, enabling the researchers to examine asymmetrical disease effects. For control subjects, bilateral averages were used for comparison. Overall, the methodological approach combined rigorous clinical assessment, comprehensive cognitive testing, and high-resolution quantitative PET imaging to evaluate the physiological and functional effects of trihexyphenidyl in early-stage Parkinson’s disease [2].

Discussion
1) The results of the study performed by the research team of Downs et al demonstrate that dopamine neurotransmission is significantly impaired in Dyt1 knock-in mice and that THP can partially restore this deficit through mechanisms involving nicotinic acetylcholine receptors (nAChRs). Using fast scan cyclic voltammetry, the researchers first established that evoked dopamine release in the dorsolateral striatum was markedly reduced in Dyt1 mice compared to wild-type controls. Specifically, dopamine concentrations following stimulation were approximately half of those observed in control animals. This reduction was also observed in the nucleus accumbens, indicating that the deficit is not restricted to motor-related regions but reflects a broader dysfunction in dopaminergic signaling. Importantly, dopamine clearance rates (tau), which reflect reuptake efficiency, were not significantly different between genotypes, suggesting that the deficit arises from impaired release rather than altered reuptake [1].
The study then examined the effects of THP on dopamine release. THP increased dopamine release in a dose-dependent manner in both control and Dyt1 mice. However, the magnitude of this effect was significantly smaller in Dyt1 mice, particularly at higher concentrations. This indicates that while THP can enhance dopamine release in the disease model, its efficacy is reduced compared to normal conditions. A similar pattern was observed with atropine, another muscarinic receptor antagonist, supporting the conclusion that the effect is mediated through muscarinic receptor blockade rather than off-target actions. In contrast, administration of L-DOPA, a precursor used to treat dopamine deficiency in other disorders, had no effect on dopamine release in either genotype. This finding aligns with the idea that dopamine synthesis is not impaired in DYT1 dystonia; instead, the problem lies in release mechanisms.
In vivo microdialysis experiments confirmed these findings under physiological conditions. Baseline extracellular dopamine levels were significantly lower in Dyt1 mice. Administration of THP, either directly into the striatum or systemically, significantly increased extracellular dopamine in both groups. Notably, THP restored dopamine levels in Dyt1 mice to values comparable to those of untreated wild-type controls, demonstrating its ability to normalize dopaminergic signaling in vivo [1].
To investigate the mechanism underlying THP’s effects, the researchers examined the role of nAChRs. Blocking nAChRs with the antagonist DHβE significantly reduced dopamine release in both genotypes and completely abolished the dopamine-enhancing effect of THP. After washout of the antagonist, THP’s effect returned, indicating that functional nAChRs are required for THP to increase dopamine release. In contrast, blocking glutamate receptors with CNQX had no effect on THP-induced dopamine release, suggesting that glutamatergic signaling is not necessary for this mechanism. This finding narrows the pathway to cholinergic modulation rather than broader excitatory input [1].
Further experiments revealed that nAChR function is altered in Dyt1 mice. Dose-response analysis with DHβE showed a leftward shift in Dyt1 mice, indicating increased sensitivity to receptor blockade. This suggests reduced baseline cholinergic tone or altered receptor function. Supporting this interpretation, experiments with acetylcholinesterase inhibitors, which increase acetylcholine levels, showed a rightward shift in dose-response curves in Dyt1 mice, indicating reduced sensitivity. However, direct measurement of AChE activity showed no difference between genotypes, implying that the changes are not due to enzyme activity but rather to altered receptor or neurotransmitter dynamics.
Additional tests examined whether nAChR function itself was fundamentally impaired. Experiments comparing tonic and burst stimulation patterns showed no difference between genotypes, suggesting that basic receptor function remains intact under these conditions. Similarly, exposure to high concentrations of nicotine, which desensitize nAChRs, produced comparable effects in both groups. Gene expression analysis also revealed no differences in nAChR subunit mRNA levels between Dyt1 and control mice [1].
Together, these findings indicate that while nAChR expression and basic responsiveness are preserved, cholinergic signaling is functionally altered in Dyt1 mice, likely due to changes in acetylcholine availability or receptor regulation. This altered cholinergic environment reduces the effectiveness of THP and contributes to impaired dopamine release. In summary, the results show that Dyt1 dystonia is associated with a significant deficit in dopamine release, that THP can restore this deficit through a mechanism dependent on nAChRs, and that abnormalities in cholinergic signaling underlie both the disease phenotype and the reduced efficacy of THP. These findings highlight nAChRs as a potential therapeutic target for improving treatment strategies in DYT1 dystonia [1].
2) The results of the study done by Takahashi et al demonstrate that treatment with trihexyphenidyl produced significant improvements in motor symptoms in patients with Parkinson’s disease, while simultaneously leading to widespread reductions in rCBF and rCMRO₂, without causing measurable cognitive decline. Clinically, all six patients exhibited improvement following treatment, as reflected in significant reductions in their UPDRS scores. The mean total UPDRS score decreased from 35.0 to 25.7, while the motor subscale score decreased from 26.4 to 19.2, both changes reaching statistical significance. These findings confirm the expected therapeutic effect of anticholinergic treatment in alleviating motor dysfunction associated with Parkinson’s disease, likely through modulation of the imbalance between cholinergic and dopaminergic systems in the basal ganglia [2].

Figure 1: Changes in rCBF and rCMRO in various brain regions both before and after treatment with THP.
Despite these motor improvements, cognitive performance remained stable throughout the study period. As shown in the neuropsychological data presented in the table on page 3 of the article, there were no significant differences between pre-treatment and post-treatment scores on any of the cognitive measures administered. Scores on the MMSE, as well as the full-scale, verbal, and performance IQ components of the WAIS-R, did not change significantly after treatment. Similarly, no significant changes were observed in any of the subscales of thevWMS-R, including measures of verbal memory, visual memory, delayed recall, and attention or concentration. Although untreated PD patients initially exhibited slightly lower performance IQ compared to healthy controls, this difference did not worsen following treatment. These results indicate that short-term administration of trihexyphenidyl does not impair cognitive function in patients with early-stage Parkinson’s disease who do not have preexisting dementia [2].
At baseline, prior to treatment, certain regional abnormalities in cerebral blood flow and metabolism were already evident in PD patients. Specifically, rCBF in the contralateral parietal cortex was significantly lower compared to controls, suggesting early disruption of cortical function associated with the disease. Additionally, rCMRO₂ in the contralateral putamen was higher than in the ipsilateral putamen, reflecting asymmetrical metabolic activity corresponding to the uneven distribution of motor symptoms. These findings are consistent with the known pathophysiology of Parkinson’s disease, in which degeneration of dopaminergic neurons leads to functional imbalances within basal ganglia circuits.
Following treatment with trihexyphenidyl, PET imaging revealed significant decreases in rCBF across multiple brain regions. On the contralateral side, reductions were observed in the striatum as well as in the frontal, parietal, and temporal cortices. On the ipsilateral side, significant decreases in rCBF were noted in the putamen and frontal cortex. The graphical data presented in Figure 2 on page 4 illustrate these changes, showing consistent reductions in blood flow across affected regions after treatment. Quantitatively, rCBF decreased by approximately 15% in the striatum and by about 10% across cortical areas. In contrast, no significant changes in rCBF were observed in the thalamus, cerebral white matter, or cerebellum, indicating that the effects of trihexyphenidyl were regionally selective rather than global [2].
Parallel reductions were observed in oxygen metabolism, as measured by rCMRO₂. On the contralateral side, significant decreases were found in the striatum and occipital cortex, while more limited changes were observed on the ipsilateral side. The data shown in Figure 3 on page 4 further support these findings, demonstrating reductions in metabolic activity following treatment. When compared with control subjects, PD patients exhibited significantly lower rCMRO₂ in the parieto-occipital cortices and the caudate nucleus after treatment, particularly on the contralateral side. These findings suggest that trihexyphenidyl suppresses neuronal metabolic activity in both cortical and subcortical regions.
Interestingly, the study found no significant changes in the rOEF before and after treatment, indicating that the observed decreases in rCBF and rCMRO₂ were not due to alterations in oxygen extraction efficiency. Instead, these changes likely reflect a reduction in neuronal activity or synaptic transmission, consistent with the inhibitory effects of anticholinergic drugs on the central nervous system. Furthermore, after treatment, rCBF and rCMRO₂ values in many brain regions were significantly lower than those observed in healthy controls, suggesting that trihexyphenidyl induces widespread suppression of cerebral function beyond the baseline abnormalities associated with Parkinson’s disease [2].
Overall, the results indicate that while trihexyphenidyl effectively improves motor symptoms in early-stage Parkinson’s disease, it also produces significant reductions in cerebral blood flow and metabolic activity across multiple brain regions, without causing detectable cognitive impairment over the short term. These findings highlight a complex relationship between cholinergic modulation, motor function, and cerebral physiology, and raise important considerations regarding the long-term effects of anticholinergic therapy on brain function [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Downs AM, Fan X, Donsante C, Jinnah HA, Hess EJ. Trihexyphenidyl rescues the deficit in dopamine neurotransmission in a mouse model of DYT1 dystonia. Neurobiol Dis. 2019;125:115-122. doi:10.1016/j.nbd.2019.01.012

[2] Takahashi S, Tohgi H, Yonezawa H, Obara S, Yamazaki E. The effect of trihexyphenidyl, an anticholinergic agent, on regional cerebral blood flow and oxygen metabolism in patients with Parkinson’s disease. J Neurol Sci. 1999;167(1):56-61. doi:10.1016/s0022-510x(99)00142-2

Trihexyphenidyl HCl is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.