Pharmacogenetic stimulation of cholinergic pedunculopontine neurons reverses motor deficits in a rat model of Parkinson’s disease

Pienaar et al. Molecular Neurodegeneration (2015) 10:47 DOI 10.1186/s13024-015-0044-5 RESEARCH ARTICLE Open Access Pharmacogenetic stimulation of cholinergic pedunculopontine neurons reverses motor deficits in a rat model of Parkinson’s disease Ilse S. Pienaar1,2*, Sarah E. Gartside3, Puneet Sharma1, Vincenzo De Paola4, Sabine Gretenkord3, Dominic Withers4, Joanna L. Elson5,6 and David T. Dexter1 Abstract Background: Patients with advanced Parkinson's disease (PD) often present with axial symptoms, including postural- and gait difficulties that respond poorly to dopaminergic agents. Although deep brain stimulation (DBS) of a highly heterogeneous brain structure, the pedunculopontine nucleus (PPN), improves such symptoms, the underlying neuronal substrate responsible for the clinical benefits remains largely unknown, thus hampering optimization of DBS interventions. Choline acetyltransferase (ChAT)::Cre+ transgenic rats were sham-lesioned or rendered parkinsonian through intranigral, unihemispheric stereotaxic administration of the ubiquitin-proteasomal system inhibitor, lactacystin, combined with designer receptors exclusively activated by designer drugs (DREADD), to activate the cholinergic neurons of the nucleus tegmenti pedunculopontine (PPTg), the rat equivalent of the human PPN. We have previously shown that the lactacystin rat model accurately reflects aspects of PD, including a partial loss of PPTg cholinergic neurons, similar to what is seen in the post-mortem brains of advanced PD patients. Results: In this manuscript, we show that transient activation of the remaining PPTg cholinergic neurons in the lactacystin rat model of PD, via peripheral administration of the cognate DREADD ligand, clozapine-N-oxide (CNO), dramatically improved motor symptoms, as was assessed by behavioral tests that measured postural instability, gait, sensorimotor integration, forelimb akinesia and general motor activity. In vivo electrophysiological recordings revealed increased spiking activity of PPTg putative cholinergic neurons during CNO-induced activation. c-Fos expression in DREADD overexpressed ChAT-immunopositive (ChAT+) neurons of the PPTg was also increased by CNO administration, consistent with upregulated neuronal activation in this defined neuronal population. Conclusions: Overall, these findings provide evidence that functional modulation of PPN cholinergic neurons alleviates parkinsonian motor symptoms. Keywords: Cholinergic, Deep brain stimulation, DREADD, Parkinson’s disease, Pedunculopontine nucleus Background The pedunculopontine nucleus (PPN), located in the dorsal tegmentum of the midbrain and upper pons, regulates aspects of cognition, sleep architecture, motivation, reward and locomotion [1, 2], potentially via ascending and descending, afferent and efferent connections to many * Correspondence: 1 Centre for Neuroinflammation and Neurodegeneration, Division of Brain Sciences, Faculty of Medicine, Imperial College London, London W12 ONN, UK 2 Department of Applied Sciences, Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, UK Full list of author information is available at the end of the article brain regions [3]. Studies reported a neuronal loss in the PPN of patients with Parkinson’s disease (PD) [4, 5] and in the most common atypical parkinsonian syndrome, progressive supranuclear palsy [4, 6]. This neuronal loss principally affects cholinergic neurons and may underlie several of the motor abnormalities seen in PD patients. Experimental evidence for this was provided by the selective destruction of the pedunculopontine cholinergic subpopulation in rats [7] and also in macaques [8], through an intra-pedunculopontine infusion of a diphtheria toxin (Dtx) conjugated to the peptide Urotensin II © 2015 Pienaar et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pienaar et al. Molecular Neurodegeneration (2015) 10:47 (UII) (Dtx::UII), serving as the endogenous ligand for urotensin-II receptors that are expressed only by cholinergic neurons in this region. Dtx::UII induces a gradual cholinergic-specific cell death by inhibiting the synthesis of choline transporter [9]. Following lesion formation in the rats, no effect was seen on execution of individual motor actions; however, impairments emerged when the demands of the task increased, i.e., during an acrobatic locomotor task (accelerating rotarod) [7]. This result mimicked that reported from the primate study, where the lesion induced no changes in the levels of baseline locomotion; however, the animals displayed significant changes in gait and posture when they were assessed in a guided and trained semibipedal walking task [8]. Although no single genetic-based/toxin-induced animal model of PD perfectly recapitulates all neuropathological characteristics and clinical symptoms of the human disorder [10], recent results have highlighted the potential of the lactacystin rat model for studying neuropathological mechanisms underlying PD [11]. In rats receiving a unilateral injection of the irreversible ubiquitin-proteasomal system inhibitor, lactacystin, into the substantia nigra pars compacta (SNc), we recently reported a loss of cholinergic neurons in the nucleus tegmenti pedunculopontine (PPTg), the rodent equivalent of the human PPN [12], which at 5 weeks following the lesion, resembles the level of neuronal loss observed in advanced human PD [11]. The toxin was shown to affect non-cholinergic neurons in this nucleus also, which mimics observations made in post-mortem PPN tissue from PD-affected patients [5, 13]. This includes recent findings [5], which revealed that alpha-synuclein (αSYN) overexpression (resembling Lewy body pathology) affects both cholinergic and non-cholinergic neurons in the PPN of post-mortem PD-affected brains as well as in the PPTg of lactacystin-lesioned rats. In line with this, a loss of both neuronal types was observed in the PD cohort, although non-cholinergic neurons were less impacted upon than the cholinergic population [5]. The pathological alterations, concomitant with nigrostriatal deficits and PD-related motor abnormalities seen in the lactacystin rat model of PD [11, 13] strongly supports the notion that a unilateral injection of lactacystin in rats represents a useful experimental tool by which to investigate the impact of PPN cholinergic deficits in PD, over that of more traditional experimental mo (...truncated)