Jennifer L. Tillerson

Exercise induces behavioral recovery and attenuates neurochemical deficits in rodent models of Parkinson's disease.

Exercise is thought to improve motor function and emotional well-being in patients with Parkinsons disease (PD). However, it is not clear if the improvements are due to neurochemical alterations within the affected nigrostriatal region or result from a more general effect of exercise on affect and motivation. In this study we show that motorized treadmill running improves the neurochemical and behavioral outcomes in two rodent models of PD: the unilateral 6-hydroxydopamine (6-OHDA) rat model and bilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model in aged C57bl mice. Exposure to the dopamine (DA) toxins 6-OHDA or MPTP resulted in permanent behavioral and neurochemical loss. In contrast, when lesioned animals were exposed to treadmill activity two times a day for the first 10 days post-lesion they displayed no behavioral deficits across testing days and had significant sparing of striatal DA, its metabolites, tyrosine hydroxylase, vesicular monoamine transporter, and DA transporter levels compared to lesion sedentary animals. These results demonstrate that exercise following nigrostriatal damage ameliorates related motor symptoms and neurochemical deficits in rodent models of PD.

Neuroprotective effects of prior limb use in 6-hydroxydopamine-treated rats: possible role of GDNF

Unilateral administration of 6‐hydroxydopamine (6‐OHDA) into the medial forebrain bundle (MFB) causes a loss of dopamine (DA) in the ipsilateral striatum and contralateral motor deficits. However, if a cast is placed on the ipsilateral limb during the first 7 days following 6‐OHDA infusion, forcing the animal to use its contralateral limb, both the behavioral and neurochemical deficits are reduced. Here, we examine the effect of forced reliance on a forelimb during the 7 days prior to ipsilateral infusion of 6‐OHDA on the deficits characteristic of this lesion model. Casted animals displayed no behavioral asymmetries as measured 14–28 days postlesion and a marked attenuation in the loss of striatal DA and its metabolites at 30 days. In addition, animals receiving a unilateral cast alone had an increase in glial cell‐line derived neurotrophic factor (GDNF) protein in the striatum corresponding to the overused limb. GDNF increased within 1 day after the onset of casting, peaked at 3 days, and returned to baseline within 7 days. These results suggest that preinjury forced limb‐use can prevent the behavioral and neurochemical deficits to the subsequent administration of 6‐OHDA and that this may be due in part to neuroprotective effects of GDNF.

Forced Nonuse in Unilateral Parkinsonian Rats Exacerbates Injury

Diagnosis of Parkinsons disease (PD) is based on the presentation of clinical symptoms such as bradykinesia, resting tremor, and rigidity. However, one feature of PD that often begins years before diagnosis is decreased physical activity. We hypothesized that this depressed activity is not only a symptom of the early dopaminergic loss but also a catalyst in the degenerative process. Two experiments were performed to test this hypothesis. First, rats were exposed to a mild dose of 6-hydroxydopamine unilaterally into the nigrostriatal dopamine (DA) projections, which would normally result in an ∼20% DA loss and no detectable behavioral asymmetries. A subset of these lesioned animals then had a cast applied for 7 d to the contralateral forelimb. After the cast was removed, these animals displayed long-term behavioral asymmetry and exacerbation of neurochemical loss (∼60% depletion). Second, a group of animals received a high dose of 6-hydroxydopamine that normally would yield a severe loss of nigrostriatal terminals (∼90% loss) and chronic sensorimotor deficits. During the first 7 d after neurotoxin exposure, a subset of these animals were forced to rely on the contralateral forelimb, a procedure we have previously reported to protect DA terminals and behavioral function. Some of these rats then had the use of their “recovered” forelimb restricted during the second or third week after lesioning. This precipitated a severe and chronic loss of DA terminals and functional deficits. These results suggest decreased physical activity not only is a symptom of PD but also may act to potentiate the underlying degeneration.

Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine

Overt behavioral symptoms of Parkinsons disease (PD) do not occur until over 80% of the striatal dopamine content has been lost. Diagnosis of the disorder relies on identifying clinical symptoms including akinesia, resting tremor, and rigidity. In retrospect, behavioral deficits are observed several years prior to diagnosis. Behavioral manifestations in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, such as changes in general locomotor activity and rotorod performance, require large doses of MPTP and are often transient. We hypothesized that, as in PD, subtle behavioral changes also occur in the MPTP model. In this paper, we demonstrate that mice treated with moderate doses of the dopaminergic toxin MPTP display deficits in behavioral parameters that are significantly correlated with the loss of striatal dopamine. In addition, these behavioral measures are correlated to dopamine transporter, vesicular monoamine transporter, and tyrosine hydroxylase expression and are improved following L-DOPA administration. Detection of dopamine-modulated behavioral changes in moderately depleted MPTP mice will allow for more efficacious use of this model in PD research.

Differential effects of glial cell line-derived neurotrophic factor (GDNF) in the striatum and substantia nigra of the aged Parkinsonian rat

Injection of an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic (DA) neurons in the substantia nigra (SN) of young rats. As Parkinson’s disease occurs primarily in aged populations, we examined whether chronic biosynthesis of GDNF, achieved by adenovirus-mediated delivery of a GDNF gene (AdGDNF), can protect DA neurons and improve DA-dependent behavioral function in aged (20 months) rats with progressive 6-OHDA lesions of the nigrostriatal projection. Furthermore, the differential effects of injecting AdGDNF either near DA cell bodies in the SN or at DA terminals in the striatum were compared. AdGDNF or control vector was injected unilaterally into either the striatum or SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the same side as the vector injection. AdGDNF injection into either the striatum or SN significantly reduced the loss of FG labelled DA neurons 5 weeks after lesion (P ⩽ 0.05). However, only striatal injections of AdGDNF protected against the development of behavioral deficits characteristic of unilateral DA depletion. Striatal AdGDNF injections also reduced tyrosine hydroxylase fiber loss and increased amphetamine-induced striatal Fos expression. These results demonstrate that increased levels of striatal, but not nigral, GDNF biosynthesis prevents DA neuronal loss and protects DA terminals from 6-OHDA-induced damage, thereby maintaining DA function in the aged rat.

Delivery of a GDNF Gene into the Substantia Nigra after a Progressive 6-OHDA Lesion Maintains Functional Nigrostriatal Connections

The effects of delivering GDNF via an adenoviral vector (AdGDNF) 1 week after lesioning dopaminergic neurons in the rat substantia nigra (SN) with 6-hydroxydopamine (6-OHDA) were examined. Rats were unilaterally lesioned by injection of 6-OHDA into the striatum, resulting in progressive degeneration of dopaminergic neurons in the SN. One week later, when substantial damage had already occurred, AdGDNF or a control vector harboring beta-galactosidase (AdLacZ) was injected into either the striatum or SN (3.2 x 10(7) PFU/microl in 2 microl). Rats were examined behaviorally with the amphetamine-induced rotation test and for forelimb use for weight-bearing movements. On day 30 postlesion, the extent of nigrostriatal tract degeneration was determined by injecting a retrograde tracer (FluoroGold) bilaterally into the lesioned striatum. Five days later, rats were sacrificed within 2 h of amphetamine injection to examine amphetamine-induced Fos expression in the striatum, a measure of dopaminergic-dependent function in target neurons. AdGDNF injection in the SN rescued dopaminergic neurons in the SN and increased the number of dopaminergic neurons that maintained a connection to the striatum, compared to rats injected with AdLacZ. Further support that these spared SN cells maintained functional connections to the striatum was evidenced by increased Fos expression in striatal target neurons and a decrease in amphetamine-induced rotation. In contrast to the effects observed in rats injected with AdGDNF in the SN, rats injected with AdGDNF in the striatum did not exhibit significant ameliorative effects. This study demonstrates that experimentally increasing levels of GDNF biosynthesis near the dopaminergic neuronal soma is effective in protecting the survival of these neurons and their function even when therapy is begun after 6-OHDA-induced degeneration has commenced. Thus, GDNF gene therapy may ameliorate the consequences of Parkinsons disease through rescuing compromised dopaminergic neurons.

Grid performance test to measure behavioral impairment in the MPTP-treated-mouse model of parkinsonism.

Behavioral impairments in mice following administration of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) require large depletions in striatal dopamine content and are often transient. In this paper, we describe a simple and inexpensive test that measures long-term behavioral deficits in mice treated with moderate doses of MPTP. These measures are significantly correlated with the loss of striatal dopamine and immunoreactivity of the dopamine transporter, vesicular monoamine transporter and tyrosine hydroxylase. In addition, behavioral impairments on the measures were reversed following L-DOPA administration. Employment of this test will allow for more efficacious use of mice in PD research, as well as provide more sensitive measures of behavioral improvement following potential therapeutic or neuroprotective interventions.

Olfactory discrimination deficits in mice lacking the dopamine transporter or the D2 dopamine receptor

Previous pharmacological studies have implicated dopamine as a modulator of olfactory bulb processing. Several disorders characterized by altered dopamine homeostasis in olfaction-related brain regions display olfactory deficits. To further characterize the role of dopamine in olfactory processing, we subjected dopamine transporter knockout mice (DAT -/-) and dopamine receptor 2 knockout mice (D2 -/-) to a battery of olfactory tests. In addition to behavioral characterization, several neurochemical markers of olfactory bulb integrity and function were examined. DAT -/- mice displayed an olfactory discrimination deficit, but did not differ detectably from DAT wildtype (DAT +/+) mice in odor habituation, olfactory sensitivity, or odor recognition memory. Neurochemically, DAT -/- mice have decreased D2 receptor staining in the periglomerular layer of the olfactory bulb and increased tyrosine hydroxylase immunoreactivity compared to DAT +/+ controls. D2 -/- mice exhibited the same olfactory deficit as the DAT -/- mice, further supporting the role of dopamine at the D2 synapse in olfactory discrimination processing. The findings presented in this paper reinforce the functional significance of dopamine and more specifically the D2 receptor in olfactory discrimination and may help explain the behavioral phenotype in the DAT and D2 knockout mice.

Glial cell line-derived neurotrophic factor (GDNF) gene delivery protects dopaminergic terminals from degeneration.

Previously, we observed that injection of an adenoviral (Ad) vector expressing glial cell line-derived neurotrophic factor (GDNF) into the striatum, but not the substantia nigra (SN), prior to a partial 6-OHDA lesion protects dopaminergic (DA) neuronal function and prevents the development of behavioral impairment in the aged rat. This suggests that striatal injection of AdGDNF maintains nigrostriatal function either by protecting DA terminals or by stimulating axonal sprouting to the denervated striatum. To distinguish between these possible mechanisms, the present study examines the effect of GDNF gene delivery on molecular markers of DA terminals and neuronal sprouting in the aged (20 month) rat brain. AdGDNF or a control vector coding for beta-galactosidase (AdLacZ) was injected unilaterally into either the striatum or the SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the side of vector injection. Two weeks postlesion, rats injected with AdGDNF into either the striatum or the SN exhibited a reduction in the area of striatal denervation and increased binding of the DA transporter ligand [(125)I]IPCIT in the lesioned striatum compared to control animals. Furthermore, injections of AdGDNF into the striatum, but not the SN, increased levels of tyrosine hydroxylase mRNA in lesioned DA neurons in the SN and prevented the development of amphetamine-induced rotational asymmetry. In contrast, the level of T1 alpha-tubulin mRNA, a marker of neuronal sprouting, was not increased in lesioned DA neurons in the SN following injection of AdGDNF either into the striatum or into the SN. These results suggest that GDNF gene delivery prior to a partial lesion ameliorates damage caused by 6-OHDA in aged rats by inhibiting the degeneration of DA terminals rather than by inducing sprouting of nigrostriatal axons.

Intervention Strategies for Degeneration of Dopamine Neurons in Parkinsonism

Parkinsonism has a complex and variable neuropathology and etiology (1,2), but the major common feature is progressive loss of dopamine (DA) neurons in the substantia nigra leading to motor impairment. Although understanding the initial causes and molecular mechanisms for degeneration of the nigrostriatal system would be invaluable for eventual treatment, in the meantime effective approaches to preventing the pathological and disabling clinical signs of the disorder may come from early detection and interventions that have quite general neuroprotective properties. Behavior-based diagnostic assessment aimed at detecting threshold level neuropathology would be critical, both in clinical practice and preclinically.