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The Intranasal Administration of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP): A New Rodent Model to Test Palliative and Neuroprotective Agents for Parkinson's disease Rui D. S. Predigera,b*, Aderbal S. Aguiar Jr. a, Eduardo L. G. Moreirab, Filipe C. Matheusa, Adalberto A. Castroc, Roger Walzb,d, Andreza F. De Bemc, Alexandra Latinib,c, Carla I. Tascab,c, Marcelo Farinab,c and Rita Raisman-Vozarie a
Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, Florianópolis-SC, Brazil, bCentro de Neurociências Aplicadas (CeNAp), Hospital Universitário, Universidade Federal de Santa Catarina, UFSC, Florianópolis, SC, Brazil, cDepartamento de Bioquímica, Universidade Federal de Santa Catarina, UFSC, Florianópolis, SC, Brazil, d Departamento de Clínica Médica, Hospital Universitário, Universidade Federal de Santa Catarina, UFSC, Florianópolis, SC, Brazil, eUMR 975 INSERM - Université Pierre et Marie Curie. Centre de Recheche de l'Institut du cerveau et de la moelle épinière CRICM Thérapeutique Expérimentale de La neurodégénérescence. Hôpital de la Salpêtrière, Paris, France Abstract: Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting approximately 1% of the population older than 60 years. Classically, PD is considered to be a motor system disease and its diagnosis is based on the presence of a set of cardinal motor signs that are consequence of a pronounced death of dopaminergic neurons in the substantia nigra pars compacta (SNc). Nowadays there is considerable evidence showing that non-dopaminergic degeneration also occurs in other brain areas which seems to be responsible for the deficits in olfactory, emotional and memory functions that precede the classical motor symptoms in PD. Dopaminereplacement therapy has dominated the treatment of PD and although the currently approved antiparkinsonian agents offer effective relief of the motor deficits, they have not been found to alleviate the non-motor features as well as the underlying dopaminergic neuron degeneration and thus drug efficacy is gradually lost. Another major limitation of chronic dopaminergic therapy is the numerous adverse effects such as dyskinesias, psychosis and behavioral disturbance. The development of new therapies in PD depends on the existence of representative animal models to facilitate the evaluation of new pharmacological agents before they are applied in clinical trials. We have recently proposed a new experimental model of PD consisting of a single intranasal (i.n.) administration of the proneurotoxin 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP, 1 mg/nostril) in rodents. Our findings demonstrated that rats and mice treated intranasally with MPTP suffer impairments in olfactory, cognitive, emotional and motor functions conceivably analogous to those observed during different stages of PD. Such infusion causes time-dependent loss of tyrosine hydroxylase in the olfactory bulb and SNc, resulting in significant dopamine depletion in different brain areas. We have also identified some pathogenic mechanisms possibly involved in the neurodegeneration induced by i.n. administration of MPTP including mitochondrial dysfunction, oxidative stress, activation of apoptotic cell death mechanisms and glutamatergic excitotoxicity. Therefore, the present review attempts to provide a comprehensive picture of the i.n. MPTP model and to highlight recent findings from our group showing its potential as a valuable rodent model for testing novel drugs that may provide alternative or adjunctive treatment for both motor and non-motor symptoms relief with a reduced side-effect profile as well as the discovery of compounds to modify the course of PD.
Keywords: Parkinson's disease, intranasal MPTP, motor symptoms, non-motor symptoms, behavior, pathogenic mechanisms, animal model, antiparkinsonian drugs screening. INTRODUCTION At the beginning of the nineteenth century, when James Parkinson first described the disorder that bears his name [1], life expectation was no longer than 45 years old. Nowadays, life expectancy is near 80 years old and the prevalence of Parkinson's disease (PD) is generally estimated at 0.3% of the entire population and about 1% in people over 60 years of age [2]. Since the incidence of the disease increases with age (the most important risk factor), it is likely that the number of people suffering from PD will rise steadily in the future. Overall, the annual economic impact of PD only in the United States is estimated at $10.8 billion, 58% of which is related to direct medical costs [3]. Classically, PD is considered to be a motor system disease and its diagnosis is based on the presence of a set of cardinal motor signs (e.g. rigidity, bradykinesia, rest tremor and postural reflex disturbance). These symptoms of PD mainly result from the progressive and profound loss of neuromelanin-containing dopaminergic neurons in the substantia nigra pars compacta (SNc) with *Address correspondence to this author at the Departamento de Farmacologia, Universidade Federal de Santa Catarina, Campus Trindade, 88049-900, Florianópolis, SC, Brazil; Tel: 55 48 3721 9491; Fax: 55 48 3337 5479; E-mail:
[email protected]
1381-6128/11 $58.00+.00
presence of eosinophillic, intracytoplasmic, proteinaceous inclusions termed as Lewy bodies and dystrophic Lewy neurites in surviving neurons [4]. Dopamine-replacement therapy has dominated the treatment of PD since the early 1960s and although the currently approved antiparkinsonian agents offer effective relief of the motor deficits, especially in the early/moderate stages of the disease, they have not been found to alleviate the underlying dopaminergic neuron degeneration and drug efficacy is gradually lost [5]. Moreover, another major limitation of chronic dopaminergic therapy is the numerous adverse effects such as the development of abnormal involuntary movements (namely dyskinesia), psychosis and behavioral disturbance (e.g., compulsive gambling, hypersexuality) [6]. The dopaminergic therapy in PD is based on the importance of nigral dopaminergic cell loss, the ensuing striatal dopamine depletion, and onset of motor symptoms. However, nowadays there is considerable evidence showing that the neurodegenerative processes that lead to sporadic PD begin many years before the appearance of the characteristic motor symptoms and additional neuronal fields and neurotransmitter systems are also involved in PD, including the anterior olfactory structures, dorsal motor nucleus of vagus, caudal raphe nuclei, locus coeruleus, the autonomic nervous system, hippocampus and the cerebral cortex [7]. Accordingly, cho© 2011 Bentham Science Publishers Ltd.
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linergic, adrenergic and serotoninergic neurons are also lost which seems to be responsible for the non-motor symptoms of PD encompassing olfactory and memory impairments, sleep abnormalities, anxiety and depression, as well as gastrointestinal disturbance, which precede the classical motor symptoms [8]. Non-motor features of PD invariably do not respond to dopaminergic medication and probably form the major current challenge faced in the clinical management of PD [8]. Therefore, the limitations of the current pharmacological treatment of PD have led to extensive investigation of novel drugs that may provide alternative or adjunctive treatment for both motor and non-motor symptoms relief with a reduced side-effect profile as well as the discovery of compounds to modify the course of PD [9,10]. The three main strategic developments that have led to progress in the medical management of PD have focused on improvements in dopaminergic therapies (including those aimed at managing or preventing the onset of motor complications), the identification of non-dopaminergic drugs for symptomatic improvement and the discovery of compounds to modify the course of PD [9,10]. The definition of neuroprotection in PD is complex and involves the potential for preventing cell death and restoring function to damaged neurons, as well as increasing neuronal number [11]. The development of drugs to slow or prevent the progression of PD might logically evolve from an improved understanding of the etiology and pathogenesis of PD. There have certainly been major advances in these areas over the past few years and the prospect for the introduction of "neuroprotective" therapies is much improved. However, despite extensive efforts and research, to date, there is no proven therapy to prevent cell death or to restore affected neurons to a normal state [12]. Preclinical studies in laboratory animals have provided several candidate neuroprotective drugs, but clinical end points are readily confounded by any symptomatic effect of the study intervention and thus do not provide an unequivocal measure of disease progression that can be used to determine if a drug has a neuroprotective effect [13,14]. The potential neuroprotective drugs include the dopamine agonists and monoamine oxidase (MAO) type B inhibitors, although others, including co-enzyme Q10, antioxidants, adenosine A2A receptor antagonists, growth factors, antiapoptotic agents and glutamate inhibitors, have also been the subjects of clinical trials in PD [9,10,12]. It can be hypothesized that the low clinical efficacy of several “neuroprotective” agents is due, at least in part, to a late diagnosis of PD. Frequently, new potential agents are tested when the patient already shows the cardinal motor signs. Unfortunately, the patients only fulfill these clinical criteria when 60-70% of the neurons of the SNc are degenerated and the striatal dopamine content is reduced by 80% [4,15]. Therefore, seems to be imperative for a better response to evaluate new candidate neuroprotective agents in early pre-motor stages of PD. The development of new neuroprotective therapies in PD depends on the existence of representative animal models to facilitate the evaluation of new pharmacological agents and therapeutic strategies before they are applied in clinical trials [16]. To date, most studies performed with animal models of PD have investigated their ability to induce nigrostriatal dopaminergic pathway damage and motor alterations associated with advanced phases of PD (for review see [17,18]). Because early preclinical stages of PD are accompanied by alterations in a variety of functions (including olfactory, cognitive and emotional) and the management of the nonmotor symptoms of PD remains a challenge [8], evaluating whether the utilized animal models of PD alter any of these functions seems important. Until recently, no well-accepted model of the early phase of PD was available in the literature. In this context, we have recently proposed a new experimental model of PD consisting of a single intranasal (i.n.) administration of the proneurotoxin 1-
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methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in rodents [1923]. As illustrated in Fig. 1, the MPTP toxicokinetics is complex and has several stages, involving inhibition of mitochondrial complex-I activity and oxidative stress [24]. Once in the brain, MPTP is rapidly converted to the toxic ion 1-methyl-4-phenyl-piperidinium (MPP+) by the enzyme MAO-B, mainly in glial cells. Unlike its precursor MPTP, MPP+ is a polar molecule and does not cross biological membranes freely. However, this toxin has a high binding affinity to the dopamine transporter (DAT), thus being captured into dopaminergic terminals [18]. Dopaminergic neurons accumulate MPP+ in mitochondria to levels that inhibit complex-I of the electron transport chain, causing a severe depletion of ATP and increased production of reactive oxygen species (ROS), particularly superoxide. The production of ROS seems to be one of the primary events of the mechanism of MPP+ neurotoxicity and, although not directly responsible for neuronal death, it acts as an activator of several cellular cascades that will cause the death of dopaminergic neurons [18,24,25]. Alternatively, MPP+ can be sequestered into cytoplasmic vesicles by actions of the vesicular monoamine transporter VMAT2, which is a proton-dependent transporter that sequesters monoamine neurotransmitters from free cytoplasmic space into synaptic vesicles. Since it structurally resembles monoamines, MPP+ can be transported by the VMAT into these vesicles, thus being prevented from entering the mitochondria (Fig. 1). This sequestration has been demonstrated to be as a potential mechanism for reducing the toxic effects of MPTP [26]. Young adult Wistar rats and C57BL/6 mice (3-6 months old) treated intranasally with MPTP suffer time-dependent impairments in olfactory, cognitive, emotional and motor functions conceivably analogous to those observed during different stages of PD. Such infusion causes loss of tyrosine hydroxylase (TH) in the olfactory bulb and SNc, resulting in significant dopamine depletion in the olfactory bulb, prefrontal cortex and striatum. We have also identified some pathogenic mechanisms possibly involved in the neurodegeneration induced by i.n. administration of MPTP including mitochondrial dysfunction, oxidative stress, activation of apoptotic cell death mechanisms and glutamatergic excitotoxicity. Therefore, the present review attempts to provide a comprehensive picture of the i.n. MPTP model and to highlight recent findings from our group showing its potential as a valuable rodent model for testing novel drugs that may provide alternative or adjunctive treatment for both motor and non-motor symptoms relief with a reduced sideeffect profile as well as the discovery of compounds to modify the course of PD. ENVIRONMENTAL TOXINS AND THE OLFACTORY VECTOR HYPOTHESIS IN PARKINSON’S DISEASE Although the etiology of the neurodegenerative process found in PD is not completely understood, currently, the most plausible explanation is that PD represents a multifactorial disease resulting from the combination of genetic and environmental factors [27,28]. The number of environmental chemicals potentially involved in the etiology of PD is staggering. On a daily basis, humans are exposed to thousands of xenobiotics in air, water, and food, including chemicals from clothing, furniture, paints, rugs, plastics, plumbing, cooking utensils, perfumes, cosmetics, personal care products, food, beverages, pesticides, herbicides, and vehicular, industrial and municipal emissions [29]. In some cases such agents may enter the brain via the olfactory neuroepithelium, a concept termed the olfactory vector hypothesis [30]. In accord with this hypothesis, a number of studies have shown that i.n. infusion of viruses [31] or cadmium [32], or inhalation of aluminum [33] or manganese [34,35], can result in the invasion of these agents into the brain, in some cases severely damaging central brain structures. Also in accord with this hypothesis are the observations that approximately 90% of patients with early-stage PD
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Fig. (1). Schematic illustration of the complex metabolism of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neurotoxicity pathway. After its administration, MPTP rapidly crosses the blood-brain barrier and is metabolized to 1-methyl-4-phenyl-2,3-dihydropyridinium (MPP+) by the enzyme monoamine oxidase B (MAO-B) in glial cells (mainly astrocytes), and then, probably by spontaneous oxidation, to 1-methyl-4-phenylpyridinium (MPP+), the active toxic compound. MPP+ is then taken up by dopamine transporter (DAT), for which it has high affinity. Once inside dopamine neurons, MPP+ is concentrated by an active process within the mitochondria, where it impairs mitochondrial respiration by inhibiting complex I of the electron transport chain, resulting in an increased production of free radicals, which causes oxidative stress and activation of programmed cell death molecular pathways. The schematic is adapted from Vila and Przedborski [24]. See text for further citations.
exhibit olfactory dysfunction unrelated to disease stage or the use of anti-PD medications [30,36]. Moreover, Braak et al. [7] presented evidence, based on pathological studies, that the earliest PD-related brain pathology occurs within the olfactory bulb and the dorsomedial nucleus of the vagus nerve. Recently, Hawkes in collaboration with Braak and colleagues [37], has proposed a “dual hit” hypothesis in which both the olfactory and vagus nerves become involved simultaneously, perhaps from a pathogen that enters the nose and becomes swallowed with the nasal secretions, passing the stomach wall into Auerbach’s and Meissner’s plexuses. This has suggested to some that a xenobiotic may enter both the nose and the gut to initiate the disease process [37]. A detailed review of the anatomy of the nose and the olfactory vector hypothesis is beyond the scope of this article and can be found elsewhere [30]. The anatomy of the nose is well suited for the transfer of exogenous agents into the brain. Although some xenobiotics, notably viruses, can enter the brain via several cranial nerves, the olfactory nerve (cranial nerve I) is uniquely vulnerable to such penetration. Thus, the dendritic knobs and protruding cilia of the 6 to 10 million olfactory receptor cells that make up this nerve provide an exposed surface area conservatively estimated at 23 cm2 [38]. These cells are widely distributed throughout the rostral nasal cavity, embedded in a specialized neuroepithelium that lines the region of the cribriform plate, the dorsal septum, and sectors of the superior and middle turbinates. Unlike other receptor cells, these
cells are also first-order neurons, projecting axons directly to the brain without an intervening synapse [30]. The connection between the nasal cavity and the central nervous system (CNS) by the olfactory neurons has been investigated extensively during the last decades with regard to its feasibility to serve as a direct drug transport route to the brain [39]. As illustrated in Fig. 2, compounds that reach the respiratory epithelium are absorbed into the systemic circulation or cleared by mucociliary clearance and swallowed. Molecules that are absorbed into the systemic circulation may enter the CNS after passing through the blood-brain barrier (BBB). When a molecule is deposited directly on the olfactory epithelium it is possible that transport via the olfactory neurons occurs. Two possible routes exist by which molecules can be transported from the olfactory epithelium into the brain and/or cerebrospinal fluid (CSF). The first is the epithelial pathway, where compounds pass paracelularly across the olfactory epithelium into the perineural spaces, crossing the cribriform plate and entering the subarachnoid space filled with CSF. From that the molecules can diffuse into the brain tissue or will be cleared by the CSF flow into the lymphatic vessels and subsequently into the systemic circulation. The second possibility is the olfactory nerve pathway, where compounds may be internalized into the olfactory neurons and pass inside the neuron through the cribriform plate into the olfactory bulb. It is possible that further transport into the brain can occur by bridging the synapses between the neurons. After
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Fig. (2). Schematic illustration of the possible passage routes of xenobiotics and drugs following intranasal administration.
reaching the brain tissue, drugs are cleared either via the CSF flow or via efflux pumps such as p-glycoprotein at the BBB into the systemic circulation [40]. Furthermore, the trigeminal nerve [41] and, in animals, the vomeronasal organ [42] also connect the nasal cavity with the brain tissue. Despite the favorable anatomy of the nose for the transfer of exogenous agents into the brain, surprisingly, few studies have addressed the possibility that neurotoxins such as MPTP could damage the basal ganglia following i.n. administration. In this context, we have recently proposed a new experimental model of PD consisting of a single i.n. administration of MPTP in rats and mice and further details of the main findings obtained using this animal model will be addressed in the next sections. THE INTRANASAL MPTP ADMINISTRATION REPRODUCES CLINICAL FEATURES OF PARKINSON’S DISEASE - RELEVANCE FOR THE SEARCH OF PALLIATIVE DRUGS PD is one of the several human diseases that seem not to occur spontaneously in other animals. However, the characteristics of this disease can be mimicked in laboratory animals through the administration of various neurotoxic agents that disturb dopaminergic neurotransmission, for example 6-hydroxydopamine (6-OHDA), MPTP and rotenone [17,18,29]. However, there are still no accepted progressive models of PD that mimic the processes known to occur during cell death and that result in the motor and nonmotor deficits, pathology, biochemistry, and drug responsiveness as seen in humans [43]. Despite these limitations, over the past couple of decades, the proneurotoxin MPTP has become a widely used approach for modeling PD. The development of this model is based on the accidental discovery made in the early 1980s, when a parkinsonian syndrome in young drug addicts in Northern California was linked to their unintentional MPTP self-administration via the intravenous injection of MPTP-contaminated “synthetic heroin” [44]. Subsequent work has revealed that MPTP is not toxic by itself, but that it easily enters the brain where it is metabolized to the active toxin MPP+ [25] (see Fig. 1). In humans and nonhuman primates, MPTP causes a severe and irreversible PD-like syndrome [44,45]. In contrast to primates, rodents are less sensitive to MPTP toxicity [46]. Nevertheless, the C57BL/6 mouse strain has been found to be sensitive to
the systemic injection of MPTP and is significantly more susceptible than other mouse strains in terms of affecting mesencephalic dopaminergic neurons [47]. Largely because of the economic, logistic, and ethical constraints related to experimental research in primates, the MPTP mouse model has become the most commonly used animal model of PD [46-48]. On the other hand, the MPTP model is not widely used in rats largely because systemic doses comparable to those used in mice do not produce significant dopaminergic degeneration [49-51]. The conspicuous insensitivity of rats to MPTP toxicity may be due to the fact that the rat brain capillaries contain exceptionally high levels of MAO-B, which constitutes an effective enzymatic BBB [50]. Therefore, our work hypothesis was that since the i.n. route bypasses the BBB and avoids peripheral metabolic effects capable of decreasing an agent’s effectiveness in getting to the brain [32], then the nasal cavity might represent an additional route for the research of MPTP effects in rats and presenting the important advantage to avoid the need of stereotactic administration of MPP+. Consistent with this suggestion, Kawano and Margolis [52] have demonstrated that the i.n. administration of 6-OHDA, a neurotoxin incapable of crossing the BBB, produces a significant reduction in the olfactory bulb noradrenaline levels of female mice. Interestingly, at the same time that our group published the first paper describing the i.n. MPTP model in rats [19], Rojo et al. [53] proposed a mouse model of PD based on chronic i.n. infusion of MPTP. After 30 days of repeated i.n. MPTP infusion (60 mg/kg/day) mice developed a significant reduction in ambulatory behavior that correlated with a drop of striatal dopamine to 20% and a drastic reduction in the TH immunoreactivity of striatum and SNpc [53]. The same group also investigated the effects of chronic (30 days) i.n. delivery of the neurotoxins rotenone and paraquat in rodents. However, in contrast to the findings obtained with MPTP, rotenone-treated mice or rats were asymptomatic while i.n. administration of paraquat induced severe hypokinesia and vestibular damage, but did not alter nigrostriatal system [54]. Although the chronic i.n. inoculation of MPTP has undoubtedly contributed to a better understanding of many features of PD, for example assessing the risk from environmental neurotoxins, this model induces nigrostriatal pathway damage and motor alterations associated with advanced phases of PD.
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As stated in the introduction, PD seems to be a multidimensional disease, and besides motor deficits, it is associated with a number of sensorial, cognitive and emotional disturbances that result in a loss in quality of life of the individuals [8]. The symptoms observed in the early preclinical phases of PD include olfactory loss [36,55], cognitive impairments [56-59], anxiety and depression [60,61]. Non-motor features of PD invariably do not respond to dopaminergic medication and probably form the major current challenge faced in the clinical management of PD [8]. However, few studies have addressed consistently these non-motor symptoms in animal models of PD. We therefore investigated the occurrence of such behavioral alterations in young adult Wistar rats (3 months old) and C57BL/6 mice (5-6 months old) over the course of one-month period after a single bilateral i.n. administration of MPTP (1 mg/nostril) (for more protocol details see [19-23]). MPTP HCl (Sigma Chemical Co., USA) was administered by i.n. route according to the procedure described by Dluzen and Kefalas [62] and modified in our laboratory [19-23]. Briefly, rats and mice were lightly anaesthetized with isoflurane 0.96% (0.75 CAM; Abbot Laboratórios do Brasil Ltda., RJ, Brazil) using a vaporizer system (SurgiVet Inc., WI, USA) and a 10-mm piece of PE-50 tubing (for rats) or a 7-mm piece of PE-10 tubing (for mice) was inserted through the nostrils and connected to a peristaltic pump set at a flow rate of 12.5 l/min. The MPTP HCl was dissolved in 0.9% NaCl (saline) at a concentration of 20 mg/ml, after which it was infused for 4 min (1 mg/nostril). The control solution consisted of saline. Animals were given a 1-min interval to regain normal respiratory function and then this procedure was repeated with infusions administered through the contralateral nostrils. As summarized in Table 1, the test battery included tests of odor discrimination, anxiety-like behavior, depressive-like behavior, learning and memory, and spontaneous locomotor activity. Table 1.
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Rodents treated intranasally with MPTP suffered timedependent impairments in olfactory, cognitive, emotional and motor functions [19-23] which appears to be correlated with different stages of the human PD, according to the Braak’s hypothesis [7]. For example, in the earlier stages of PD, dopaminergic neurons in the SNc are moderately lost (40-70%) and sensory and memory deficits are present in the absence of major motor impairment. As in patients with PD, the i.n. administration of MPTP promoted an early disruption in the olfactory discrimination ability of adult rats [19,21] and mice [22], verified 1-14 days after MPTP treatment. The inability of MPTP-treated animals to discriminate between the familiar and the non-familiar compartments really reflects a deficit in olfactory discrimination, and not a simple locomotor impairment, since no alteration in the open field parameters was observed at initial times (1-7 days) after MPTP treatment [19,21,23]. Since the i.n. administration of MPTP does not cause, at least at initial periods (7-10 days), gross motor alterations that would preclude assessment of cognitive and emotional functions, we also investigated whether such behaviors are affected in these animals. While the i.n. MPTP animals performed normally in the step-down inhibitory avoidance task [22] and in the spatial reference memory version of the water maze [19,21], they were significantly impaired in their ability to locate the cued platform in the water maze and impaired ability to recognize the juvenile after a short time (30 min) [19,21-23]. This implies a disruption in habit learning, as well as poor performance in working memory, consistent with the cognitive deficits observed in PD patients [56-59]. Depressive disorders commonly occur in PD [60], affecting approximately 40% of the patients during the early stages of the disease [63]. Several studies suggest that the pathophysiology underlying mood disorders in PD may be different from the mechanisms that account for the behavioral symptoms observed in the
Summary of the Olfactory, Cognitive, Emotional and Motor Symptoms Observed in the Intranasal (i.n.) MPTP Rodent Model of Parkinson's Disease
Behavioral Tests
Days after i.n. MPTP
Subjects
Results
Braak’s Hypothesis
References
Stage 1
[19,21]
Olfactory
1-14
Rats
Olfactory deficits
discrimination
5
Mice
Olfactory deficits
Social
6
Rats
Social memory deficits
Recognition
12
Mice
Social memory deficits
Water maze
7-10
Rats
Working memory deficits
Working memory
12-15
Mice
Working memory deficits
Water maze
7-10
Rats
Implicit memory deficits
Stage 2
[19,21]
7-10
Rats
Not altered by i.n. MPTP
Stage 2
[19,21]
7
Mice
Not altered by i.n. MPTP
Stage 2
[22]
16
Rats
Depressive-like behavior
Stage 2
[22]
1-7
Rats
Not altered by i.n. MPTP
Stages 1-2
[19,21,23]
14 and 21
Rats
Motor impairments
Stages 3-4
[19,21]
Activity
32
Rats
Motor impairments
Stages 3-4
[23]
chamber
18
Mice
Not altered by i.n. MPTP
Stages 3-4
[22]
[22] Stage 2
[19,21,23] [22]
Stage 2
[19,21] [22]
Procedural memory Water maze Reference memory Elevated Plus Maze Forced Swimming Open field
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general population [64]. Although the pathophysiology of psychiatric symptoms in PD is not fully understood, striatal, frontal and limbic dopaminergic, cholinergic, serotonergic, noradrenergic and GABAergic pathways are thought to be involved in their genesis [65]. The i.n. infusion of MPTP caused a depressive-like behavior in rats [23], reflected by an increased immobility time in the forced swimming test [66]. It is important to emphasize that the i.n. administration of the same MPTP dose did not alter the motor performance of rats when they were tested in the rotarod and grasping stretch tests at the same time after i.n. MPTP administration (data not published). Thus, brain structures (e.g., olfactory bulb, amygdala and locus coeruleus) implicated in sensory-motivational responses may have been affected by i.n. MPTP, and the present increase in the immobility time in the forced swimming test is not directly related to motor abnormalities. Moreover, since up to 40% of patients with PD suffer from clinically significant anxiety [65], we evaluated whether anxiogenic-like responses may also occur in the present i.n. MPTP model. The elevated plus-maze test was used on the basis of its documented ability to detect both anxiolytic- and anxiogenic-like drug effects in mice [67]. However, no significant alteration in anxiety-related parameters (i.e., time and number of entries in the open arms) evaluated in the elevated plus-maze was observed in MPTP-treated mice [22] and rats (unpublished data). Contrasting with the earlier findings obtained in rats [19,21,23], the single i.n. administration of MPTP did not alter the motor performance of mice evaluated in activity chambers, at least until 18 days after treatment [22]. Therefore, it is important to emphasize that the replication of clinical features of PD in MPTP rodent models seems to be dependent of many variables, such as species, strain, gender, age and/or the schedule of treatment utilized. In fact, the recovery of motor performance appears to be almost universal following acute and sub-acute MPTP models [17,68]. On the other hand, the chronic MPTP administration over several weeks by continuous infusion [69], or in association with probenecid [68,70], was shown to induce a persistent degeneration of nigrostriatal neurons associated with long-lasting motor deficits in mice. It should be pointed out, however, that our PD rodent model does not fully mimic human PD. Thus, despite the fact that the observed sequence of olfactory, cognitive, emotional and motor impairments is similar to the sequence of analogous changes seen in PD, the time frame of development of these problems is shorter in our i.n. MPTP rodent model and the olfactory dysfunction reverses itself. Such reversal might not occur in older animals, something that requires additional study.
Table 2.
With the purpose of determining the relationship between the behavioral alterations observed in rodents infused with a single i.n. administration of MPTP and neurochemical alterations in monoaminergic neurotransmission, the levels of dopamine, 3,4dihydroxyphenylacetic acid (DOPAC), homovallinic acid (HVA), noradrenaline, and 5-hydroxytryptamine (5-HT) were assayed by high performance liquid chromatography (HPLC) in the olfactory bulb, striatum, prefrontal cortex and hippocampus. As illustrated in Table 2, the olfactory, cognitive, emotional and motor deficits observed in the present i.n. MPTP model were correlated with a marked dopamine depletion in the olfactory bulb, striatum, and prefrontal cortex, but not hippocampus, of MPTPtreated rodents. The levels of dopamine metabolites DOPAC and HVA were also significantly decreased in these brain structures (data not shown). Additionally, noradrenaline concentration was decreased in the olfactory bulb, striatum and hippocampus, whereas no significant alterations of noradrenaline were seen in prefrontal cortex. Moreover, no significant differences in the 5-HT levels were observed in any of the brain structures investigated (Table 2). Figure 3 shows representative photomicrographs of TH immunohistochemistry in the ventral mesencephalom and striatum at 20 days after i.n. administration of MPTP or control solution (saline). As can be seen, immunohistochemistry revealed a pronounced loss of TH-positive neurons in SNc (70% loss) of MPTP-treated mice. Moreover, optical density measurements demonstrated that the i.n. administration of MPTP induced a significant reduction of TH immunostaining in the striatum (80% lower) (Fig. 3). At this point, it is important to emphasize that rats are significantly less susceptible than mice in terms of affecting mesencephalic dopaminergic neurons after the i.n. MPTP infusion, displaying only a moderate loss of TH-positive neurons in SNc (about 35-40% loss) [19,23]. Of high importance, additional histological analysis using Nissl staining revealed a cellular loss in the SNc of MPTP-treated mice [22], indicating that the observed reduction of TH-positive cells is associated to dopamine neurons degeneration, thus discarding a possible TH downregulation in absence of neuronal death. Therefore, despite the existence of some limitations, the finding that MPTP introduced intranasally to mice and rats more closely mimics the development of clinical features of PD (including nonmotor and motor symptoms, SNc cell death and depletion of dopamine levels in different brain areas) than MPTP introduced via other routes [19-23], is in accord with the concept that some forms of PD may be caused by xenobiotics that enter the brain via the olfactory pathways [29]. Interestingly, in agreement with our experimental data, there is one case report of a chronic parkinsonism
The Effects of Intranasal Administration of MPTP (1 mg/nostril) on Dopamine, Noradrenaline and 5-hydroxytryptamine Levels in Different Brain Areas of Mice
Treatment
BRAIN AREA
Dopamine
Noradrenaline
5-hydroxytryptamine
control
Olfactory bulb
354.22+22.02
268.34+24.51
481.10+53.36
267.76+15.51*
205.30+6.56*
503.11+56.01
14281.34+1531.95
91.59+14.05
426.70+65.20
2324.21+301.40*
49.71+7.58*
400.72+43.15
MPTP control
Striatum
MPTP control
Prefrontal
1171.92+222.56
293.66+22.63
445.39+39.02
MPTP
cortex
670.06+105.00*
306.51+13.84
543.80+57.12
control
Hippocampus
MPTP
21.79+4.93
287.18+12.38
523.08+21.10
14.88+3.70
229.23+10.79*
552.00+45.00
The values represent the mean ± S.E.M. monoamine levels (ng/g wet tissue) of 4–8 animals in each group. *P
