Parkinson's Disease (PD) is the second-most prevalent neurodegenerative disorder in the United States, affecting as many as one million people (NINDS, 2018). The number of diagnoses is expected to rise as the population of aged persons grows, threatening to impact a greater majority of the population (Kowal et al., 2013; Marras et al., 2018). This disease is both chronic and progressive, so by the time a diagnosis is made, patients have already progressed to the point where they struggle to control their movements (NINDS, 2018).

PD is characterized by progressive dopamine (DA) neuron death in the Substantia Nigra pars compacta (SNc; Ehringer & Hornykiewicz, 1998) in addition to concomitant aggregations of misfolded α-synuclein proteins termed Lewy Bodies (Davie, 2008), resulting in the cardinal symptoms of PD: rigidity, tremor, postural instability, bradykinesia, and akinesia (Jankovic, 2008). Inflammation also plays a crucial role in the etiology of PD, suggesting an involvement of reactive astrocytes and reactive microglia in the SNc (McGeer & McGeer, 2008, Mosley et al., 2006, Tansey et al., 2006). 

Due to the plasticity of the surrounding neurons and the ability of serotonin (5-hydroxytryptamine; 5-HT) neurons to produce and release DA, symptoms do not begin to present themselves until about 80% of dopaminergic projections to the striatum have died. At this point, 5-HT neurons, which lack the DA transporter (DAT) responsible for reuptaking and recycling DA, begin to lose control of the delicate balance between the direct (excitatory) striatonigral and indirect (inhibitory) striatopallidal pathways of the basal ganglia, suppressing brainstem efferents and thereby reducing motor activity. As these symptoms progress, walking, talking, swallowing, and other simple tasks can become very challenging for patients (NINDS, 2018).

Though motor symptoms are often the focus of PD research, numerous non-motor symptoms plague PD patient including cognitive impairment, mood and behavioral problems, sleep disorders, and constipation. Furthermore, some of these symptoms appear long before motor impairments arise, sometimes allowing for earlier diagnosis of PD. Hyposmia, characterized by the reduced ability to detect orders, REM sleep-behavior disorder, characterized by acting out vivid dreams, and constipation will typically precede motor symptoms by several years (NINDS, 2018). Additional early symptoms can include anxiety, depression(Winter et al., 2007), excessive sweating and digestive and/or respiratory issues(Brooks & Doder, 2001).  After the development of motor symptoms, many PD patients will develop dementia as well. While treatments have been developed to ameliorate the symptoms of PD, there is no treatment that can slow its progression (Smith et al., 2011).

As loss of dopaminergic tone leads to the symptoms of PD, DA replacement therapy (DRT) is used to provide exogenous DA via L-3,4-dihydroxyphenylalanine (L-DOPA) pills at regular intervals. L-DOPA, the "gold standard" pharmacotherapy, is the precursor to DA, and is capable of crossing the blood brain barrier (BBB), whereas DA cannot pass the BBB itself. Once in the parkinsonian brain, L-DOPA is uptaken by 5-HT neurons and surviving DA neurons, decarboxylated into DA, and is released from axon terminals in the striatum. At first this treatment successfully ameliorates akinesia and drastically improves quality of life in a majority of patients (Birkmayer & Hornykiewicz, 1961). However, the drug is not without it's downsides - some studies suggest the L-DOPA may be toxic to the few surviving DA neurons in the SNc of patients, though results are inconclusive(Olanow et al., 2004; ELLDOPA Study, 2004), and as many as 90% of patients will develop Abnormal Involuntary Movements (AIMs), known as L-DOPA-Induced Dyskinesia (LID), within the first decade of treatment (Ahlskog & Muenter, 2001). 

LID is most commonly characterized by rapid involuntary movements, or chorea, as well as sustained muscle contractions, or dystonia (Thanvi et al., 2007). While there is no known cause as to why some patients and animals will become dyskinetic but others remain resistant. Some studies suggest that LID results from a loss of bidirectional striatal synaptic plasticity(Picconi et al., 2003), while many other studies suggest this effect is caused, at least in part, by the large fluctuations in plasma concentrations of L-DOPA and consequent pulsatile DA stimulation that result from the L-DOPA ingestion intervals. Some studies suggest this ectopic over-release of DA in the SNc increased susceptibility of SN neurons to L-DOPA-induced neurotoxicity(Mosharov et al., 2009). Over time, LID greatly narrows the therapeutic window (Jankovic, 2005), therefore increasing the economic burden of PD (Suh et al., 2012) and reducing the quality of life for patients (Chapuis et al., 2005; Manson et al., 2012). While some have suggested using DA agonists to delay the onset of LID (Schwarz, 2003), our lab has shown that LID emerges regardless of DA agonist or L-DOPA treatment(Lanza et al., 2019). Despite the downsides of DRT, a vast majority of patients prefer the dyskinestic side effects over akinetic parkinsonism, greatly underscoring the need for research into the reduction of LID.

Treatment success is vastly different in patients with advanced-stage PD, as they may experience a wearing-off of the beneficial effects of PD drugs. In these situations, L-DOPA can induce further motor dysfunction like postural imbalance, freezing of gait, and falls. Some patients will also suffer from behavioral changes including heightened stress(Macht et al., 2015), impulse control disorders, hallucinations, and psychosis.  (NINDS, 2018). In severe and treatment-resistant cases of PD, Deep Brain Stimulation (DBS) is sometimes employed, as it can reduce tremor, rigidity, stiffness, and can improve movement, though its mechanism of action has not been deciphered (NINDS, 2018). Though there are numerous therapies to ameliorate the symptoms of PD, none have demonstrated the ability to slow the progression of PD (Poewe, 2009; NINDS, 2018).

As the exact causes of PD are unknown, numerous rat models are employed to recapitulate different neurodegenerative aspects of PD. Some models an involve infusions of toxins, while others focus on genetic modifications mimicking deleterious mutations sometimes found in PD patients. Toxin models can involve the chronic or acute administration of a neurotoxin administered either locally or systemically, and include 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP), rotenone, or paraquat (Bové et al., 2005). Genetic models include modifications to genes encoding α-synuclein, leucine-rich repeat kinase 2 (LRRK2), Parkin, DJ-1, and PTEN-induced kinase 1 (PINK1). Such modifications mimic the protein and mitochondiral dysfunction seen in PD patients. Additional models may involve the tissue-specific knockdown of a particular gene product.

6-Hydroxydopamine 

Bishop most frequently employs a unilateral 6-hydroxydopamine (6-OHDA) neurotoxin model, as opposed to bilateral lesions (Paillè  et al., 2007), as unilateral lesions provide a within-subjects control group. In this model, an acute dose of the toxin is infused directly into the medial forebrain bundle, upon which the 6-OHDA is mistaken for L-DOPA and is uptaken by nearby DA neurons. The neuron's attempt to produce DA from 6-OHDA results in the formation of reactive oxygen species, leading to death of as many as 95-99% of dopaminergic neurons residing in the SNc (Deumens et al., 2002; Przedborski & Ischiropoulos, 2005; BISHOP PAPER). This highly-efficacious and minimally-invasive methodology has been adapted from previous research which involved numerous injections of 6-OHDA across the striatum (Kirik et al., 1998). Further modifications have involved the use of graded lesions to mimic early, mid, and late-stage PD (Truong et al., 2006). While a reliable model, it is important to keep in mind that the 6-OHDA lesion model's effects are not limited to DA in the SNc - numerous other neurotransmitters and neuromodulators are impacted, and changes in electrophysiology and energy consumption within affected cells may occur (Schwarting & Huston, 1996).

1-Methyl-4-phenyl-1,2,3,6- tetrahydropyridine

The MPTP model is frequently employed in non-human primates, as it allows a gradual progression of PD. In this model, 1-methyl-4-phenylpyridinium (MPP+) is infused either locally or systemically. MPP+ crosses the BBB, after which Monoamine Oxidase B converts it to toxic metabolite MPTP.  Local infusions of MPP+ into the medial forebrain bundle produced DA loss and behavioral aberrations similar to nigral lesions caused by 6-OHDA (Sindhu et al., 2005).

Rotenone

Rotenone and MPTP both produce a parkinsonian phenotype by acting as mitochondrial neurotoxins (Sindhu et al., 2005), thus mimicking mitochondrial dysfunction found in some PD patients (NINDS, 2018). Some have demonstrated the emergence of α-synuclein- and poly-ubiquitin-positive aggregates in the DA neurons of the SNc, recapitulating the Lewy Bodies found in PD patients (Cannon et al., 2009).

A number of behavioral tests are employed to monitor the effects of PD and L-DOPA treatment across a variety of measures ranging from dyskinesia to motor impairment/improvement to anxiety and depression and even startle response.

Forepaw Adjustment Stepping (FAS) Test

The FAS test is an effective proxy for approximating lesion severity and for measuring changes in motor impairment following the administration of antiparkinsonian drugs (Chang et al., 1999). Animals displaying ≥ 75% deficits in stepping of the lesioned forepaw often demonstrate ≥ 95% DA denervation in the SNc following a 6-OHDA lesion to the MFB. For this test, an animal is held so that its hind limbs and one forelimb are restrained. The animal is held so that its remaining free forepaw rests on a table, where it is dragged laterally 90cm in 10 sec. The number of steps the rat takes is recorded and then this process is repeated in the forehand and backhand direction three times each, and is again repeated with the other paw. Animals may undergo this test to measure motor impairment, therefore approximating lesion success. Rats displaying more effective lesions often step less than their unlesioned counterparts. Such information is often used to create testing groups of equally-disabled rats. Following injection of L-DOPA or other antiparkinsonian drug, this test may again be employed to measure improvements in motor behavior. 

Abnormal Involuntary Movements (AIMs) Test

The AIMs test has been proven reliable for quantifying the severity of LID (Lundblad et al., 2002; Carta et al., 2006). Before the start of the test, rats will often be injected with a dyskinetogenic drug like L-DOPA, sometimes in conjunction with an anti-dyskinetic drug, and placed in a clear plexiglass cylinder for the duration of the testing period. The expression of dyskinesia is then rated according to three main dyskinetic sub-types: 

• axial - characterized by twisting of the torso contralateral to lesion, 

• limb - characterized by punching/flailing and clenching motions crossing the midline of the body, and 

• oralingual - characterized by side-to-side jaw movements and tongue protrusions. 

Ratings occur for 1 minute every 10 minutes over a 180 minute period. Each behavior is rated from 0-4: 

• 0 meaning the behavior is not present, 

• 1 signifying the behavior is present for <30 sec, 

• 2 signifying the behavior is present for ≥ 30 sec, 

• 3 signifying the behavior is present for the entire 60 sec, but can be interrupted by an external stimulus (tap on cylinder), and 

• 4 signifying the behavior is present for the entire duration and can not be interrupted by a stimulus. 

Rotations are also counted during the rating period, where clockwise 360° rotations are considered positive turns, and counterclockwise 360° rotations are considered negative turns. Locomotor score is assigned based on the number of rotations:

• 1 if the rat completes between 1 and 7 positive rotations, and 

• 2 if the rat completes rotates 8 or more rotations.

If you want to learn more, check out the following articles recommended by Bishop Lab:

Birkmayer and Hornykiewicz, 1961

Bjorklund and Dunnett, 2007 (DA Review)

Borah and Mohanakumar, 2007 (L-DOPA and 5-HT)

Bove et al., 2005 (Toxin-induced models of PD)

Brooks and Doder, 2001 (Depression in PD)

Buck and Ferger, 2009 (NE and AIMs)

Calabresi et al., 2008 (LID mechanisms)

Cannon et al., 2009 (Rotenone model)

Carta et al., 2006 (AIMs Models)

Carta et al., 2007 (5-HT terminals and LID)

Chang et al., 1999 (FAS)

Delaville et al., 2011 (NE and PD)

Deumens et al., 2002 (6-OHDA lesion)

ELLDOPA Study, 2004

Gerfen et al., 2008 (ERK and D1)

Graybiel et al., 1990 (c-fos and DA)

Iravani et al., 2006 (+DPAT and primate)

Kirik et al., 1998 (Intrastriatal 6-OHDA)

Kuhn and Arthur, 1998 (5-HT and quinones)

Kuhn and Arthur, 1999 (L-DOPA and TPH)

Lundblad et al., 2002 (AIMs test)

Macht et al., 2005 (Stress and PD)

McGreer and McGreer, 2008 (PD and Glia)

Morgante et al., 2006 (Motor Ctx and LID)

Mosharov et al., 2009 (L-DOPA and toxicity)

Mosley et al., 2006 (Inflammation and PD)

Nutt et al., 2010 (LID and AP response)

Olanow et al., 2004- L-DOPA+Toxicity

Paille et al., 2007 (Bilateral 6-OHDA)

Picconi et al., 2003 (Plasticity in LID)

Poewe, 2009 (PD overview)

Pum et al., 2009 (PFC 5-HT and Anxiety)

Putterman et al., 2007 (AIMs and DA)

Robelet et al., 2004 (LID and glutamate)

Robertson et al., 1989 (c-fos and D1)

Santini et al., 2009 (LID and ERK)

Schallert et al., 2000 (Cylinder test etc.)

Scholtissen et al., 2006 (5-HT in PD)

Schwarting and Huston, 1996 (6-OHDA model)

Schwarz, 2003 (DA monotherapy)

Scott and Aperia, 2009 (NMDA and D1)

Sindhu et al., 2005 (Rotenone and behavior)

Smith et al., 2011 (L-DOPA challenges)

Tansey et al., 2007 (Triggers, Inflammation and PD)

Terzioglu and Galter, 2008 (Models of PD)

Thomas and Huganir, 2004 (MAPK Review)

Truong et al., 2006 (Graded Lesions)

Westin et al., 2007 (ERK and striatum)

Winter et al., 2007.  (SNc and VTA lesions and Depression)

Yelnick, 2002 (Functional Neuroanatomy of BG)

Zhang et al., 2008 (5-HT1B)