Alterations in primary motor cortex neurotransmission and gene expression in hemi-parkinsonian rats with drug-induced dyskinesia
Introduction
Parkinson’s disease (PD) is principally caused by the loss of dopamine (DA) cells in the substantia nigra, leading to poverty of movement (Dauer and Przedborski, 2003, Jankovic, 2008). Treatment with l-DOPA relieves PD symptoms, but long-term use typically causes l-DOPA-induced dyskinesias (LID) that are in part due to supersensitization of DA D1 receptors (Cenci et al., 2011, Feyder et al., 2011). An alternative strategy to treating PD has involved the use of primary motor cortex (M1) transcranial magnetic stimulation, which has shown promise in two meta-analyses (Fregni et al., 2005, Elahi et al., 2009).
Even though conventional anti-PD therapies modulate M1 activity and the region can be directly targeted for symptomatic relief, relatively little is known about how M1 monoamine transmission and gene expression are altered in human PD patients or in associated animal models (Lindenbach and Bishop, 2013). In the lone post-mortem study of M1 catecholamine fibers in PD patients, axons staining positively for tyrosine hydroxylase (TH; predominantly DA neurons: Hokfelt et al., 1977, Miner et al., 2006) were reduced by 24–74% compared to controls, depending on the cortical layer (Gaspar et al., 1991). In the popular 6-hydroxydopamine (6-OHDA) rat model of PD, reductions in M1 TH-positive fibers have been reported using optical density (Halje et al., 2012) or qualitative histology (Debeir et al., 2005), but there have been no attempts to rigorously quantify the extent of fiber loss. Changes in M1 monoamine tissue concentrations have not been assessed in humans or rat models of PD. Studies in parkinsonian primates have sometimes reported reductions in M1 DA, norepinephrine (NE) and serotonin (5-HT) levels, while other studies have found M1 monoamines to be equal to controls despite severe subcortical monoamine pathology (Pifl et al., 1991, Engeln et al., 2015). It is unclear how these changes in monoamine innervation effect cellular physiology in M1, although, at least in the prefrontal cortex, DA receptors modulate both glutamate and GABA currents (Lewis and O’Donnell, 2000, Seamans et al., 2001a, Seamans et al., 2001b). A similar pattern may be occurring in M1, as animal models of PD typically show abnormal firing patterns of M1 glutamatergic and GABAergic cells (Watts and Mandir, 1992, Parr-Brownlie and Hyland, 2005, Pasquereau and Turner, 2011, Brazhnik et al., 2012, Halje et al., 2012).
The influence of DA depletion and exogenous DA replacement on local M1 gene expression is unclear, although a key role for M1 DA is to facilitate motor learning, likely through promoting plasticity in M1 (Floel et al., 2005, Hosp and Luft, 2013). Under normal circumstances, motor learning in M1 is associated with DA-dependent induction of the immediate-early gene c-Fos: expression levels rise while learning a motor task and decline nearly to control levels with repeated performance of the task (Kleim et al., 1996, Hosp et al., 2011). Since LID is often viewed as a pathological form of motor learning that is coincident with striatal c-Fos induction, it is possible that M1 c-Fos is involved in abnormal motor learning during LID (Calabresi et al., 2000, Calabresi et al., 2015, Mura et al., 2002). While multiple laboratories have reported that M1 c-Fos is induced by l-DOPA during dyskinesia (Ostock et al., 2011, Halje et al., 2012), these studies have been performed only in animals with multiple exposures to l-DOPA and the contribution of D1 receptors to this effect is unclear. Whereas c-Fos is critical for affecting transcriptional activity, another immediate-early gene, activity-regulated cytoskeletal-associated protein (ARC), is important for promoting synaptic plasticity in part through AMPA receptor trafficking, and may identify unique aspects of cortical plasticity (Bramham et al., 2008, Korb and Finkbeiner, 2011, Perez-Cadahia et al., 2011). Indeed, ARC protein was recently shown to be preferentially enhanced by l-DOPA among rats that displayed significant LID as opposed to more stable l-DOPA responders (Bastide et al., 2014).
The goal of the present study was to characterize structural and functional changes occurring in M1 in a widely used rat model of PD/LID, in order to spur further research and highlight therapeutic approaches. First, 6-OHDA-induced changes in M1 TH-fiber innervation and monoamine tissue concentrations were quantified using immunohistochemistry and high performance liquid chromatography (HPLC). Next, real-time reverse-transcriptase polymerase chain reaction (PCR) was used to examine changes in M1 gene expression after DA lesion and treatment with l-DOPA or the D1 receptor agonist SKF81297 (SKF). Our hypothesis was that 6-OHDA would reduce DA and NE innervation of M1, while DA replacement would pathologically enhance expression of M1 immediate-early genes and other genes involved in glutamate signaling, coincident with the induction of dyskinetic behavior.
Section snippets
Animals
All experiments used male Sprague–Dawley rats (Taconic Farms, Hudson, NY, USA) that were 9–11 weeks old at the start of the experiment (N = 86). Rats were pair-housed in plastic cages and given free access to water and standard laboratory rat food. The colony room was maintained at 22–23 °C on a 12-h light/dark cycle, with experiments taking place during the light cycle. Throughout the study, animals were cared for in full accordance with the guidelines of the Institutional Animal Care and Use
Experiment 1: Effect of lesion on striatal and M1 TH fiber innervation
This experiment examined the effect of 6-OHDA lesion on TH fiber innervation in the striatum (using optical density) and in M1 (using stereology). The behavioral phenotype of Parkinsonism was verified with the FAS test; rats with a 6-OHDA lesion averaged 43% intact stepping and took significantly fewer steps with their affected forelimb than sham-lesioned rats (t10 = 8.86, p < .001). Effects on TH-positive fibers were determined with a 2×2 mixed-model ANOVA: Hemisphere (Ipsilateral or Contralateral
Summary of findings
For the first time, functional and structural changes in M1 were characterized using the popular unilateral 6-OHDA rat model of PD. Behavioral aspects of the model were validated by showing that rats manifested a PD motor impairment and developed dyskinesia upon exposure to l-DOPA or the D1 agonist SKF (Figs. 3A, B, 6A). 6-OHDA caused significant TH fiber loss in M1, but did so exclusively in the hemisphere ipsilateral to lesion (Fig. 2C–E). In lesioned animals, M1 tissue concentrations of DA
Conclusions
This is the first study to characterize changes in M1 monoamine innervation and gene expression using the 6-OHDA rat model of PD and drug-induced dyskinesia. Results show that 6-OHDA pathologically reduces catecholamine fibers, although compensatory plasticity may be sufficient to maintain relatively normal DA levels even if NE levels are coincidentally suppressed. Administration of l-DOPA or a D1 receptor agonist strongly induced two immediate-early genes in M1 involved in plasticity,
Author contributions
Designed Research: DL, MMC, CYO, KBD, CB.
Performed Research: DL, MMC, CYO, KBD.
Analyzed Data: DL, MMC, CYO, KBD, CB.
Wrote Manuscript: DL, CB.
Provided Funding: KBD, CB.
Acknowledgements
The authors wish to thank Jessica A. George and Dr. Karen L. Eskow Jaunarajs for assistance with chromatography and animal testing. Thanks also to Dr. Terrence Deak, Dr. Lisa M. Savage and Dr. Caryl E. Sortwell for providing critical commentary on the manuscript.
This work was supported by National Institutes of Health grants R01-NS059600 (CB) and F31-NS066684 (KBD) as well as the Center for Development and Behavioral Neuroscience at Binghamton University. The authors declare that they have no
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