Aging accentuates decrease in tyrosine hydroxylase immunoreactivity associated with the increase in the motor impairment in a model of reserpine-induced parkinsonism.

Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by progressive dopaminergic neuron loss. Animal models have been used to develop a better understanding of the pathophysiologic mechanisms of PD. However, these models are usually conducted with young animals diverging of the age of PD patients, suggesting a bias in translational science. Thus, the aim of the study was to evaluate the effect of the age on rats in a progressive parkinsonism model induced by reserpine (RES). Adult (6 - 8 month-old) or elderly (18 - 24 month-old) male rats were assigned to six groups: control-elderly (CTL-ELDERLY), reserpine-elderly (RES-ELDERLY), reserpine-elderly withdrawal (RES-ELDERLY WITHDRAWAL), control-adult (CTL-ADULT), reserpine-adult (RES-ADULT), and reserpine-adult withdrawal (RES-ADULT WITHDRAWAL). Animals received 15 injections every other day of RES (0.1 mg / kg) or vehicle during 30 days. Throughout treatment, animals were evaluated in the catalepsy test (every 48 h) and open field test (24 h after the second injection), and weight assessment (every 4 days) was also made. Upon completion of behavioral tests, rat brains were collected for tyrosine hydroxylase (TH) immunohistochemical analysis. Main results demonstrated that RES-treated animals spent more time in the catalepsy bar compared with control groups, moreover the RES-elderly group showed a longer catalepsy time compared with the RES-ADULT group. A shorter time from RES treatment to the development of symptoms was observed in the RES-ADULT group, compared with the RES-ELDERLY group. In addition, RES-induced weight loss in both RES-ELDERLY and RES-ADULT when compared with their corresponding controls. Cessation of RES treatment was followed by weight gain only in the RES-ADULT group. A significant decrease in TH-immunoreactive cells was observed in the substantia nigra pars compacta (SNpc) and dorsal striatum (STR) in the rats in both the RES-ADULT and RES-ELDERLY groups and in the ventral tegmental area in rats in the RES-ADULT group. Furthermore, TH immunoreactivity decrease was not reversible in SNpc and STR in the RES-ELDERLY. These results show that RES has an age-dependent effect in rats, suggesting a greater sensitivity of the dopaminergic pathway to RES with advancing age. These suggest that the RES rat model of parkinsonism can be useful in improving our knowledge on the effect of aging on neurodegeneration.

Testosterone propionate improves motor alterations and dopaminergic damage in the reserpine-induced progressive model of Parkinson's disease.

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder with a higher susceptibility to occur in men. Studies suggest that this susceptibility is related to the hormonal differences observed between men and women, being a risk factor for PD. In addition, testosterone supplementation has shown controversial results in animal models of PD and parkinsonian patients. This study evaluated the effect of chronic administration of testosterone propionate (TP) on motor behavior and neurochemical parameters in the reserpine-induced rat model of parkinsonism. Male Wistar rats received 15 injections of reserpine (RES - 0.1 mg/kg) every other day and were concomitantly treated with different doses (0.1, 1.0, or 5.0 mg/kg) of daily TP for 30 days. The rats were euthanized 48 h after the 15th injection of RES or vehicle. Brains were removed and subjected to Tyrosine hydroxylase (TH) immunohistochemistry. TP at 1.0 mg/kg reduced the damages caused by reserpine in the vacuous chewing and tong protrusion behaviors and prevented dopaminergic damage in the SNpc, VTA, and Striatum. TP at 5.0 mg/kg reduced the damages caused by reserpine in the catalepsy and tong protrusion behaviors, prevented the weight loss, and prevented dopaminergic damage in the VTA. Our results suggest that chronic administration of TP has a protective effect in a rat model of parkinsonism, improving motor alterations and dopamine depletion induced by RES.

Motor, memory, and anxiety-like behavioral impairments associated with brain-derived neurotrophic factor and dopaminergic imbalance after inhalational exposure to deltamethrin.

Believed to cause damage to the nervous system and possibly being associated with neurodegenerative diseases, deltamethrin (DM) is a type II pyrethroid used in pest control, public health, home environment, and vector control. The objective of this study was to evaluate the motor, cognitive and emotional changes associated with dopaminergic and BDNF imbalance after DM exposure in rats. Sixty Wistar rats (9-10 months-old) were used, under Ethics Committee on Animal Research license (ID 19/2017). The animals were randomly divided into four groups: control (CTL, 0.9% saline), DM2 (2 mg DM in 1.6 mL 0.9% saline), DM4 (4 mg of DM in 1.6 mL of 0.9% saline), and DM8 (8 mg of DM in 1.6 mL of 0.9% saline). DM groups were submitted to 9 or 15 inhalations, one every 48 h. Half of the animals from each group were randomly selected and perfused 24 h after the 9th or 15th inhalation. Throughout the experiment, the animal's behavior were evaluated using catalepsy test, open field, hole-board test, Modified Elevated Plus Maze, and social interaction. At the end of the experiments, the rats were perfused transcardially and their brains were processed for Tyrosine Hydroxylase (TH) and Brain derived neurotrophic factor (BDNF) immunohistochemistries. The animals submitted to 9 inhalations of DM showed a reduction in immunoreactivity for TH in the Substantia nigra pars compacta (SNpc), ventral tegmental area (VTA), and dorsal striatum (DS) areas, and an increase in BDNF in the DS and CA1, CA3 and dentate gyrus (DG) hippocampal areas. Conversely, the animals submitted to 15 inhalations of DM showed immunoreactivity reduced for TH in the SNpc and VTA, and an increase in BDNF in the hippocampal areas (CA3 and DG). Our results indicate that the DM inhalation at different periods induce motor and cognitive impairments in rats. Such alterations were accompanied by dopaminergic system damage and a possible dysfunction on synaptic plasticity.

Nociception alterations precede motor symptoms in a progressive model of parkinsonism induced by reserpine in middle-aged rats.

Nociception alterations are frequent non-motor symptoms of the prodromal phase of Parkinson's disease (PD). The period for the onset of symptoms and the pathophysiological mechanisms underlying these alterations remain unclear. We investigated the course of nociception alterations in a progressive model of parkinsonism induced by reserpine (RES) in rats. Male Wistar rats (6-7 months) received 5 or 10 subcutaneous injections of RES (0.1 mg/kg) or vehicle daily for 20 days. Motor evaluation and nociceptive assessment were performed throughout the treatment. At the end of the treatment rats were euthanized, the brains removed and processed for immunohistochemical analysis (TH and c-Fos). The RES-treated rats exhibited an increased nociceptive response to mechanical and chemical stimulation in the electronic von Frey and formalin tests, respectively. Moreover, these alterations preceded the motor impairment observed in the catalepsy test. In addition, the RES treatment reduced the TH-immunoreactivity in the ventral tegmental area (VTA) and increased the c-Fos expression in the ventral-lateral periaqueductal gray (vlPAG), rostral ventral medulla (RVM) and dorsal raphe nucleus (DRN) after noxious stimuli induced by formalin. Taken together, our results reinforce that nociceptive changes are one of the early signs of PD and monoamine depletion in basal ganglia can be involved in the abnormal processing of nociceptive information in PD.

Balance alterations and reduction of pedunculopontine cholinergic neurons in early stages of parkinsonism in middle-aged rats.

The purpose of the present study was to investigate balance alterations and the possible role of the cholinergic neurons in the pedunculopontine nucleus (PPN) in the early stages of a progressive animal model of Parkinson's disease (PD). Twenty-eight middle-aged (8-9 months) male Wistar rats received 4 or 10 subcutaneous vehicle (control, CTL) or reserpine (RES) injections (0.1 mg/kg). The animals were submitted to different behavioral tests. Forty-eight hours after the 4th injection, half of the animals of each group (n = 7) were perfused and submitted to immunohistochemical analysis for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT). The remaining animals (n = 7 per group) were killed 48 h after the 10th injection. RES group presented motor deficits in the catalepsy and open field tests starting at days 12 and 20 of treatment, respectively (only for the animals that received 10 injections). On the other hand, dynamic and static balance changes were observed at earlier stages of RES treatment, starting at days 6 and 4, respectively. At this point of the treatment, there was no decrease in the number of TH immunoreactivity neurons in the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and dorsal striatum (DS). However, a decrease was observed in SNpc and dorsal striatum of animals that received 10 injections. In contrast, there was a decrease in the number of ChAT immunoreactive cells in PPN concomitantly to the balance alterations at the early stages of treatment (after 4 RES injections). Thus, by mimicking the progressiveness of PD, the reserpine model made it possible to identify static and dynamic balance impairments prior to the motor alterations in the catalepsy and open field tests. In addition, changes in balance were accompanied by a reduction in the number of ChAT immunoreactive cells in NPP in the early stages of treatment.

Short-term but not long-term exposure to an enriched environment facilitates the extinction of aversive memory.

Environmental enrichment (EE) has been used to investigate behavioral changes and neuroplasticity in brain in normal and pathological conditions. Besides, the EE has been used to understand the neurobehavioral systems involved in learning experiences, visual inputs, defensive responses, social interactions and memory. However, the required exposure duration to remove aversive memories remains lacking. Therefore, the purpose of the present study was to investigate the time-course effect of EE exposure on the extinction of aversive memory. Young adult male Wistar rats were exposed to two different EE protocols: short-term environmental enrichment (EE2 - animal kept under enriched conditions for two weeks) and long-term environmental enrichment (EE4 - animal kept under enriched conditions for four weeks). The contextual fear conditioning test was used to assess aversive memory. The both EE protocols provide changes in Zif-268 immunoreactivity in mesocorticolimbic areas such as CA1 and central amygdala; however, only short-term EE reduces the ZIF-268 immunoreactivity in VTA. Besides, both EE protocols also provide an increase in TH immunoreactivity in VTA and nucleus accumbens, but only the short-term EE modifies the TH immunoreactivity in CA1 and infralimbic region of the prefrontal cortex. The time-course effect of EE interferes differently on the extinction of aversive memory, being two weeks of exposure with EE sufficient to cause improvement in coping during aversive situations, favoring the extinction of conditioned fear memory.

Intracerebroventricular injection of deltamethrin increases locomotion activity and causes spatial working memory and dopaminergic pathway impairment in rats.

Deltamethrin (DM) is widely used in agriculture, veterinary medicine and control of domestic pests. Epidemiological studies suggest that DM exposure is a risk factor for neurodegenerative disorders such as Parkinson's (PD) and Alzheimer diseases; however the mechanisms are elusive. In the present study we evaluated the effects of intracerebroventricular (i.c.v.) administration of DM on locomotion activity, spatial working memory and dopaminergic pathway in the rat. Middle-aged male Wistar rats received three i.c.v. injections of DM 0.5 µg, DM 5 µg or vehicle, every other day. Across the treatment, the animals were submitted to behavioral evaluation in the catalepsy test, open field test, and spontaneous alternation task. Following completion of behavioral tests, rats were perfused and their brains were processed to tyrosine hydroxylase (TH) immunohistochemistry. We observed that i.c.v. administration of DM 5 µg increased locomotion activity (open field) and caused spatial working memory impairment (spontaneous alternation task). These alterations were accompanied by reduction TH immunoreactivity in the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and dorsal striatum. Conversely, no motor change was observed in the catalepsy test. These results indicate that i.c.v. administration of DM can cause hyperactivity and cognitive alteration which may be related to disruption of the dopaminergic pathway.

Sex differences in the progressive model of parkinsonism induced by reserpine in rats.

Parkinson's disease (PD) exhibits sexual differences in susceptibility and pathogenesis in humans, with a high incidence in men and a high severity of motor symptoms in male rodents. Furthermore, studies showed that the administration of low dose of reserpine (RES) induces a progressive appearance of motor alterations similar with parkinsonism in male rodents. Here, we investigated sex differences in motor deficits and tyrosine hydroxylase (TH) immunoreactivity induced by a progressive model of parkinsonism. Gonadally intact male and female Wistar rats and ovariectomized female rats received 15 subcutaneous injections (s.c.) every other day of 0.1 mg/kg of RES or vehicle. The repeated administration of low doses of RES (0.1 mg/kg) produces sexually dimorphic impairments on motor performance (catalepsy and open field test). Intact and ovariectomized females were more resistant to the deleterious effect of repeated administration of reserpine in the early, but this resistance in intact female disappears over time. However, intact females showed a reduction of the TH immunoreactivity in substantia nigra pars compacta, but not in ventral tegmental area and dorsal striatum. These results suggest a possible application of this model in the study of sexual dimorphism throughout the progression of PD.

Deltamethrin Intranasal administration induces memory, emotional and tyrosine hydroxylase immunoreactivity alterations in rats.

Parkinson's disease (PD) is a neurodegenerative disease related to the dopaminergic system. The etiology is not fully understood, but it is known that PD is a multifactorial disease that involves genetic and environmental factors, including pesticides. One of these, Deltamethrin (DM), has been widely used for vector control in crops, farming, veterinary medicine and domestic pest control. The purpose of the present study was to investigate the effect of DM repeated administration on motor, cognitive and emotional behavior and dopaminergic parameters. Male Wistar rats received 3 intranasal (i.n.) injections of 100 µL (50 µL/nostril) of DM 0.5 µg/µl or Vehicle (saline solution 0.9%), one injection per week. We observed that DM caused memory (novel object recognition task) and emotion (contextual conditioned fear) alterations accompanied by reduction of TH immunoreactivity in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA), and increase of TH immunoreactivity in the dorsal striatum. Motor alterations (catalepsy and open field task) were not observed throughout treatment. These findings suggest a possible early disruption of the dopaminergic pathway caused by repeated DM exposure, similar to that observed in early stages of PD.

RNA Interference of Human α-Synuclein in Mouse.

α-Synuclein is postulated to play a key role in the pathogenesis of Parkinson's disease (PD). Aggregates of α-synuclein contribute to neurodegeneration and cell death in humans and in mouse models of PD. Here, we use virally mediated RNA interference to knockdown human α-synuclein in mice. We used an siRNA design algorithm to identify eight siRNA sequences with minimal off-targeting potential. One RNA-interference sequence (miSyn4) showed maximal protein knockdown potential in vitro. We then designed AAV vectors expressing miSyn4 and injected them into the mouse substantia nigra. miSyn4 was robustly expressed and did not detectably change dopamine neurons, glial proliferation, or mouse behavior. We then injected AAV2-miSyn4 into Thy1-hSNCA mice over expressing α-synuclein and found decreased human α-synuclein (hSNCA) in both midbrain and cortex. In separate mice, co-injection of AAV2-hSNCA and AAV2-miSyn4 demonstrated decreased hSNCA expression and rescue of hSNCA-mediated behavioral deficits. These data suggest that virally mediated RNA interference can knockdown hSNCA in vivo, which could be helpful for future therapies targeting human α-synuclein.