Neurotoxicity Research

, Volume 34, Issue 1, pp 16–31 | Cite as

Temporal-Spatial Profiling of Pedunculopontine Galanin-Cholinergic Neurons in the Lactacystin Rat Model of Parkinson’s Disease

  • Joanna L. Elson
  • Rafael Kochaj
  • Richard Reynolds
  • Ilse S. Pienaar


Parkinson’s disease (PD) is conventionally seen as resulting from single-system neurodegeneration affecting nigrostriatal dopaminergic neurons. However, accumulating evidence indicates multi-system degeneration and neurotransmitter deficiencies, including cholinergic neurons which degenerate in a brainstem nucleus, the pedunculopontine nucleus (PPN), resulting in motor and cognitive impairments. The neuropeptide galanin can inhibit cholinergic transmission, while being upregulated in degenerating brain regions associated with cognitive decline. Here we determined the temporal-spatial profile of progressive expression of endogenous galanin within degenerating cholinergic neurons, across the rostro-caudal axis of the PPN, by utilizing the lactacystin-induced rat model of PD. First, we show progressive neuronal death affecting nigral dopaminergic and PPN cholinergic neurons, reflecting that seen in PD patients, to facilitate use of this model for assessing the therapeutic potential of bioactive peptides. Next, stereological analyses of the lesioned brain hemisphere found that the number of PPN cholinergic neurons expressing galanin increased by 11%, compared to sham-lesioned controls, and increasing by a further 5% as the neurodegenerative process evolved. Galanin upregulation within cholinergic PPN neurons was most prevalent closest to the intra-nigral lesion site, suggesting that galanin upregulation in such neurons adapt intrinsically to neurodegeneration, to possibly neuroprotect. This is the first report on the extent and pattern of galanin expression in cholinergic neurons across distinct PPN subregions in both the intact rat CNS and lactacystin-lesioned rats. The findings pave the way for future work to target galanin signaling in the PPN, to determine the extent to which upregulated galanin expression could offer a viable treatment strategy for ameliorating PD symptoms associated with cholinergic degeneration.


Cholinergic co-expression Galanin Lactacystin Parkinson’s disease Pedunculopontine nucleus 





Alzheimer’s disease


amino acid



area of interest


B-cell lymphoma 2


Bcl-2-associated X


choline acetyltransferase


Cresyl fast violet






deep brain stimulation


dementia with Lewy bodies






galanin [gene]


galanin receptor 2


gamma-aminobutyric acid


G-protein coupled receptors






laterodorsal tegmental nucleus


Lewy body disorders


nucleus basalis of Meynert




Parkinson’s disease


PD with dementia


pedunculopontine nucleus


phosphate-buffered saline


room temperature


substantia nigra pars compacta


substantia nigra pars reticulata




tris-buffered saline


tyrosine hydroxylase


ventral tegmental area



This study received grant support from the British Pharmacological Society and the Rosetrees Trust, awarded to ISP.

Compliance with ethical standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. Alexandris A, Liu AK, Chang RC, Pearce RK, Gentleman SM (2015) Differential expression of galanin in the cholinergic basal forebrain of patients with Lewy body disorders. Acta Neuropathol Commun 3(1):77. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Baquet ZC, Williams D, Brody J, Smeyne RJ (2009) A comparison of model-based (2D) and design-based (3D) stereological methods for estimating cell number in the substantia nigra pars compacta (SNpc) of the C57BL/6J mouse. Neuroscience 161(4):1082–1090. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Baxter MG, Bucci DJ, Gorman LK, Wiley RG, Gallagher M (2013) Selective immunotoxic lesions of basal forebrain cholinergic cells: effects on learning and memory in rats. Behav Neurosci 127(5):619–627. CrossRefPubMedGoogle Scholar
  4. Beal MF, MacGarvey U, Swartz KJ (1990) Galanin immunoreactivity is increased in the nucleus basalis of Meynert in Alzheimer’s disease. Ann Neurol 28(2):157–161. CrossRefPubMedGoogle Scholar
  5. Bentea E, Verbruggen L, Massie A (2017) The proteasome inhibition model of Parkinson’s disease. J Parkinsons Dis 7(1):31–63. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bowser R, Kordower JH, Mufson EJ (1997) A confocal microscopic analysis of galaninergic hyper-innervation of cholinergic basal forebrain neurons in Alzheimer’s disease. Brain Pathol 7(2):723–730. CrossRefPubMedGoogle Scholar
  7. Branchek TA, Smith KE, Gerald C, Walker MW (2000) Galanin receptor subtypes. Trends Pharmacol Sci 21(3):109–117. CrossRefPubMedGoogle Scholar
  8. Chan-Palay V (1988) Galanin hyperinnervates surviving neurons of the human basal nucleus of Meynert in dementias of Alzheimer’s and Parkinson’s disease: a hypothesis for the role of galanin in accentuating cholinergic dysfunction in dementia. J Comp Neurol 273(4):543–557. CrossRefPubMedGoogle Scholar
  9. Cordero-Llana O, Rinaldi F, Brennan PA, Wynick D, Caldwell MA (2014) Galanin promotes neuronal differentiation from neural progenitor cells in vitro and contributes to the generation of new olfactory neurons in the adult mouse brain. Exp Neurol 256:93–104. CrossRefPubMedGoogle Scholar
  10. Counts SE, Chen EY, Che S, Ikonomovic MD, Wuu J, Ginsberg SD, Dekosky ST, Mufson EJ (2006) Galanin fiber hypertrophy within the cholinergic nucleus basalis during the progression of Alzheimer’s disease. Dement Geriatr Cogn Disord 21(4):205–214. CrossRefPubMedGoogle Scholar
  11. Crawley JN, Wenk GL (1989) Co-existence of galanin and acetylcholine: is galanin involved in memory processes and dementia? Trends Neurosci 12(8):278–282. CrossRefPubMedGoogle Scholar
  12. Datta S, Siwek DF (2002) Single cell activity patterns of pedunculopontine tegmentum neurons across the sleep-wake cycle in the freely moving rats. J Neurosci Res 70(4):611–621. CrossRefPubMedGoogle Scholar
  13. Daubner SC, Le T, Wang S (2011) Tyrosine hydroxylase and regulation of dopamine synthesis. Arch Biochem Biophys 508(1):1–12. CrossRefPubMedGoogle Scholar
  14. Ding X, MacTavish D, Kar S, Jhamandas JH (2006) Galanin attenuates beta-amyloid (Abeta) toxicity in rat cholinergic basal forebrain neurons. Neurobiol Dis 21(2):413–420. CrossRefPubMedGoogle Scholar
  15. Elliot-Hunt CR, Holmes FE, Hartley DM, Perez S, Mufson EJ, Wynick D (2011) Endogenous galanin protects mouse hippocampal neurons against amyloid toxicity in vitro via activation of galanin receptor-2. J Alzheimers Dis 25:455–462CrossRefGoogle Scholar
  16. Elson JL, Yates A, Pienaar IS (2016) Pedunculopontine cell loss and protein aggregation direct microglia activation in parkinsonian rats. Brain Struct Funct 221(4):2319–2341. CrossRefPubMedGoogle Scholar
  17. Faure JB, Maques-Carneiro JE, Akimana G, Cosquer B, Ferrandon A, Herbeaux K, Koning E, Barbelivien A, Nehlig A, Cassel JC (2014) Attention and executive functions in a rat model of chronic epilepsy. Epilepsia 55(5):644–653. CrossRefPubMedGoogle Scholar
  18. Ferraye MU, Debu B, Fraix V, Goetz L, Ardouin C, Yelnik J, Henry-Lagrange C, Seigneuret E, Piallat B, Krack P, le Bas JF, Benabid AL, Chabardes S, Pollak P (2010) Effects of pedunculopontine nucleus area stimulation on gait disorders in Parkinson's disease. Brain 133(1):205–214. CrossRefPubMedGoogle Scholar
  19. Fort P, Luppi PH, Jouver M (1993) Glycine immunoreactive neurons in the cat brain stem reticular formation. Neuroreport 4(9):1123–1126PubMedGoogle Scholar
  20. Gai WP, Blumbergs PC, Geffen LB, Blessing WW (1993) Galanin-containing fibers innervate substance P-containing neurons in the pedunculopontine tegmental nucleus in humans. Brain Res 618(1):135–141. CrossRefPubMedGoogle Scholar
  21. Gentleman SM, Falkai P, Bogerts B, Herrero MT, Polak JM, Roberts GW (1989) Distribution of galanin-like immunoreactivity in the human brain. Brain Res 505(2):311–315. CrossRefPubMedGoogle Scholar
  22. Giehl K, Mestres P (1995) Somatostatin-mRNA expression in brainstem projections into the medial preoptic nucleus. Exp Brain Res 103(3):344–354CrossRefPubMedGoogle Scholar
  23. Glick SD, Weaver LM, Meibach RC (1981) Amphetamine enhancement of reward asymmetry. Psychopharmacology 73(4):323–327. CrossRefPubMedGoogle Scholar
  24. Harrison IF, Crum WR, Vernon AC, Dexter DT (2015) Neurorestoration induced by the HDAC inhibitor sodium valproate in the lactacystin model of Parkinson’s is associated with histone acetylation and up-regulation of neurotrophic factors. Br J Pharmacol 172(16):4200–4215. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Hirsch EC, Graybiel AM, Duyckaerts C, Javoy-Agid F (1987) Tegmental loss in the pedunculopontine tegmental in Parkinson disease and in progressive supranuclear palsy. Proc Natl Acad Sci U S A 84(16):5976–5780. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Hobson JA, Pace-Schott EF (2002) The cognitive neuroscience of sleep: neuronal systems consciousness and learning. Nat Rev Neurosci 3(9):679–693. CrossRefPubMedGoogle Scholar
  27. Kordower JH, Le HK, Mufson EJ (1992) Galanin immunoreactivity in the primate central nervous system. J Comp Neurol 319(4):479–500. CrossRefPubMedGoogle Scholar
  28. Lang-Rollin I, Maniati M, Jabado O, Vekrellis K, Papantonis S, Rideout HJ, Stefanis L (2005) Apoptosis and the conformational change of Bax induced by proteasomal inhibition of PC12 cells are inhibited by bcl-xL and bcl-2. Apoptosis 10(4):809–820. CrossRefPubMedGoogle Scholar
  29. Le Maître E, Barde SS, Palkovits M, Diaz-Heijtz R, Hökfelt TG (2013) Distinct features of neurotransmitter systems in the human brain with focus on the galanin system in locus coeruleus and dorsal raphe. Proc Natl Acad Sci U S A 110(6):E536–E545. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Li Y, Gao H, Wang Y, Dai C (2015) Investigation the mechanism of the apoptosis induced by lactacystin in gastric cancer cells. Tumour Biol 36(5):3465–3470. CrossRefPubMedGoogle Scholar
  31. Lipton MA (1946) Mechanism of the enzymatic synthesis of acetylcholine. Fed Proc 5(1 Pt 2):145PubMedGoogle Scholar
  32. Mackey S, Jing Y, Flores J, Dinelle K, Doudet J (2013) Direct intranigral administration of an ubiquitin proteasome system inhibitor in rat: behavior, positron emission tomography, immunohistochemistry. Exp Neurol 247:19–24. CrossRefPubMedGoogle Scholar
  33. Mazzone P, Lozano A, Stanzione P, Galati S, Scarnati E, Peppe A, Stefani A (2005) Implantation of human pedunculopontine nucleus: a safe and clinically relevant target in Parkinson's disease. Neuroreport 16(17):1877–1881. CrossRefPubMedGoogle Scholar
  34. McNaught KSP, Bjorklund LM, Belizaire R, Isacson O, Jenner P, Olanow CW (2002) Proteasome inhibition causes nigral degeneration with inclusion bodies in rats. Neuroreport 13(11):1437–1441. CrossRefPubMedGoogle Scholar
  35. Melander T, Hökfelt T, Rökaeus A (1986) Distribution of galanin-like immunoreactivity in the rat central nervous system. J Comp Neurol 248(4):475–517. CrossRefPubMedGoogle Scholar
  36. Mena-Segovia J, Micklem BR, Ungless MA, Bolam JP (2009) GABAergic neuron distribution in the pedunculopontine nucleus defines functional subterritories. J Comp Neurol 515(4):397–408. CrossRefPubMedGoogle Scholar
  37. Mesulam MM, Mufson EJ, Wainer BH, Levey AI (1983) Central cholinergic pathways in the rat: an overview based on an alternative nomenclature Ch1-Ch6. Neuroscience 10(4):1185–1201. CrossRefPubMedGoogle Scholar
  38. Mesulam MM, Geula C, Bothwell MA, Hersh LB (1989) Human reticular formation: cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei and some cytochemical comparisons to forebrain cholinergic neurons. J Comp Neurol 283(4):611–633. CrossRefPubMedGoogle Scholar
  39. Mineff EM, Popratiloff A, Romansky R, Kazakos V, Kaimaktschieff V, Usunoff KG, Ovtscharoff W, Marani E (1998) Evidence for a possible glycinergic inhibitory neurotransmission in the midbrain and rostral pons of the rat studied by gethyrin. Arch Physiol Biochem 106(3):210–220. CrossRefPubMedGoogle Scholar
  40. Moro E, Hamani C, Poon Y-Y, Al-Khairallah T, Dostrovsky JO, Hutchison WD, Lozano AM (2010) Unilateral pedunculopontine stimulation improves falls in Parkinson's disease. Brain 133(1):215–224. CrossRefPubMedGoogle Scholar
  41. Mrak RE, Griffin WST (2007) Dementia with Lewy bodies: definition, diagnosis, and pathogenic relationship to Alzheimer’s disease. Neuropsychiatr Dis Treat 3(5):619–625PubMedPubMedCentralGoogle Scholar
  42. Müller ML, Bohnen NI (2013) Cholinergic dysfunction in Parkinson’s disease. Curr Neurol Neurosci Rep 13(9):377. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Nair-Roberts RG, Chatelain-Badie SD, Benson E, White-Cooper H, Bolam JP, Ungless MA (2008) Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat. Neuroscience 152(4):1024–1031. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Ogren SO, Kuteeva E, Elvander-Tottie E, Hökfelt T (2010) Neuropeptides in learning and memory processes with focus on galanin. Eur J Pharmacol 626(1):9–17. CrossRefPubMedGoogle Scholar
  45. Okada K, Nishizawa K, Kobayashi T, Sakata S, Kobayashi K (2015) Distinct roles of basal forebrain cholinergic neurons in spatial and object recognition memory. Sci Rep 5(1):13158. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Oliveira VC, Carrara RC, Simoes DL, Saggioro FP, Carlotti CG Jr, Covas DT, Neder L (2010) Sudan black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections. Histol Histopathol 25(8):1017–1024.  10.14670/HH-25.1017 PubMedCrossRefGoogle Scholar
  47. Oorschot DE (1998) Total number of neurons in the neostriatal, pallidal, subthalamic, and substantia nigral nuclei of the rat basal ganglia: a stereological study using the cavalieri and optical disector methods. J Comp Neurol 366:580–599CrossRefGoogle Scholar
  48. Orlowski RZ (1999) The role of the ubiquitin-proteasome pathway in apoptosis. Cell Death Differ 6(4):303–313. CrossRefPubMedGoogle Scholar
  49. Paxinos G, Watson C (2009) The rat brain in stereotaxic coordinates. Elsevier Academic Press, San DiegoGoogle Scholar
  50. Pienaar IS, Van de Berg W (2013) A non-cholinergic neuronal loss in the pedunculopontine nucleus of toxin-evoked parkinsonian rats. Exp Neurol 248:213–223. CrossRefPubMedGoogle Scholar
  51. Pienaar IS, Elson JL, Racca C, Nelson G, Turnbull DM, Morris CM (2013) Mitochondrial abnormality associates with type-specific neuronal loss and cell morphology changes in the pedunculopontine nucleus in Parkinson disease. Am J Pathol 183(6):1826–1840. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Pienaar IS, Harrison IF, Elson JL, Bury A, Woll P, Simon AK, Dexter DT (2015a) An animal model mimicking pedunculopontine nucleus cholinergic degeneration in Parkinson’s disease. Brain Struct Funct 220(1):479–500. CrossRefPubMedGoogle Scholar
  53. Pienaar IS, Gartside SE, Sharma P, De Paola V, Gretenkord S, Withers D, Elson JL, Dexter DT (2015b) Pharmacogenetic stimulation of cholinergic pedunculopontine neurons reverses motor deficits in a rat model of Parkinson’s disease. Mol Neurodegener 10(1):47. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Pienaar IS, Lee CH, Elson JL, McGuinness L, Gentleman SM, Kalaria EN, Dexter DT (2015c) Deep-brain stimulation associates with improved microvascular integrity in the subthalamic nucleus in Parkinson's disease. Neurobiol Dis 74:392–405. CrossRefPubMedGoogle Scholar
  55. Pienaar IS, Vernon A, Winn P (2016) The cellular diversity of the pedunculopontine nucleus: relevance to behavior in health and aspects of Parkinson’s disease. Neuroscientist 23(4):415–431.
  56. Pirondi S, Giuliani A, Del VG, Giardino L, Hokfelt T, Calza L (2010) The galanin receptor 2/3 agonist Gal2-11 protects the SN56 cells against beta-amyloid 25–35 toxicity. J Neurosci Res 88(5):1064–1073. PubMedCrossRefGoogle Scholar
  57. Plaha P, Gill SS (2005) Bilateral deep brain stimulation of the pedunculopontine nucleus for Parkinson's disease. Neuroreport 16(17):1883–1887. CrossRefPubMedGoogle Scholar
  58. Pope RJP, Holmes FE, Kerr NC, Wynick D (2010) Characterisation of the nociceptive phenotype of suppressible galanin overexpressing transgenic mice. Mol Pain 6:67. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Rinne JO, Ma SY, Lee MS, Collan Y, Röyttä M (2008) Loss of cholinergic neurons in the pedunculopontine nucleus in Parkinson’s disease is related to the disability of the patients. Parkinsonism Relat Disord 14(7):553–557. CrossRefPubMedGoogle Scholar
  60. Robinson TE, Becker JB (1982) Behavioral sensitization is accompanied by an enhancement in amphetamine-stimulated dopamine release from striatal tissue in vitro. Eur J Pharmacol 85(2):253–254. CrossRefPubMedGoogle Scholar
  61. Salio C, Lossi L, Ferrini F, Merighi A (2006) Neuropeptides as synaptic transmitters. Cell Tissue Res 326(2):583–598. CrossRefPubMedGoogle Scholar
  62. Skofitsch G, Jacobowitz DM (1985) Immunohistochemical mapping of galanin-like neurons in the rat central nervous system. Peptides 6(3):509–546. CrossRefPubMedGoogle Scholar
  63. Steiner RA, Hohmann JG, Holmes A, Wrenn CC, Cadd G, Jureus A, Clifton DK, Luo M, Gutshall M, Ma SY, Mufson EJ, Crawley JN (2001) Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer’s disease. Proc Natl Acad Sci U S A 98:4194–4189CrossRefGoogle Scholar
  64. Tatemoto K, Rökaeus Å, Jörnvall H, McDonald TJ, Mutt V (1983) Galanin: a novel biologically active peptide from porcine intestine. FEBS Lett 164(1):124–128. CrossRefPubMedGoogle Scholar
  65. Taylor CL, Kozak R, Latimer MP, Winn P (2004) Effects of changing reward on performance of the delayed spatial win-shift radial maze task in pedunculopontine tegmental nucleus lesioned rats. Behav Brain Res 153(2):431–438. CrossRefPubMedGoogle Scholar
  66. Vernon A, Johansson S, Modo M (2010) Non-invasive evaluation of nigrostriatal neuropathology in a proteasome inhibitor rodent model of Parkinson's disease. BMC Neurosci 11(1):1. CrossRefPubMedPubMedCentralGoogle Scholar
  67. Vrontakis ME (2002) Galanin: a biologically active peptide. Curr Drug Targets CNS Neurol Disord 1(6):531–541. CrossRefPubMedGoogle Scholar
  68. West MJ, Slomianka L, Gundersen HJ (1991) Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231(4):482–497. CrossRefPubMedGoogle Scholar
  69. Wraith DC, Pope R, Blutzkueven H, Holder H, Vanderplank P, Lowrey P, Day MJ, Gundlach AL (2009) A role for galanin in human and experimental inflammatory demyelination. Proc Natl Acad Sci U S A 106(36):15466–15471. CrossRefPubMedPubMedCentralGoogle Scholar
  70. Zhang L, Yu W, Schroedter I, Kong J, Vrontakis M (2012) Galanin transgenic mice with elevated circulating galanin levels alleviate demyelination in a cuprizone-induced MS mouse model. PLoS One 7(3):e33901. CrossRefPubMedPubMedCentralGoogle Scholar
  71. Zhou HY, Tan YY, Wang ZQ, Wang G, Lu GQ, Chen SD (2010) Proteasome inhibitor lactacystin induces cholinergic degeneration. Can J Neurol Sci 37(02):229–234. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  • Joanna L. Elson
    • 1
    • 2
  • Rafael Kochaj
    • 3
  • Richard Reynolds
    • 4
  • Ilse S. Pienaar
    • 4
    • 5
  1. 1.Institute of Genetic MedicineNewcastle UniversityNewcastle upon TyneUK
  2. 2.Centre for Human MetabolomicsNorth-West UniversityPotchefstroomSouth Africa
  3. 3.Wolfson Centre for Age-Related Diseases, King’s College London, Guys CampusLondonUK
  4. 4.Centre for Neuroinflammation and Neurodegeneration, Division of Brain Sciences, Faculty of Medicine, Imperial College LondonLondonUK
  5. 5.School of Life SciencesUniversity of SussexBrightonUK

Personalised recommendations