~ Pergamon 0306-4522(95)00595-1 Neuroscience Vol. 72, No. 4, pp. 1023-1036, 1996 Elsevier ScienceLtd Copyright © 1996 IBRO Printed in Great Britain. All rights reserved 0306-4522/96 $15.00 + 0.00 REDUCTION IN ENKEPHALIN AND SUBSTANCE P MESSENGER RNA IN THE STRIATUM OF EARLY GRADE HUNTINGTON'S DISEASE: A DETAILED CELLULAR IN SITU HYBRIDIZATION STUDY S. J. AUGOOD,*f R. L. M. FAULL,~ D. R. LOVE§ and P. C. EMSON* *MRC Molecular Neuroscience Group, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, U.K. :~Department of Anatomy and §School of Biological Science, University of Auckland, Private Bag 92019, Auckland, New Zealand Abstract--The expression of enkephalin and substance P messenger RNAs was examined in the caudate-putamen of human post mortem tissue from control and Huntington's disease tissue using in situ hybridization techniques and human specific enkephalin and substance P [35S]oligonucleotides. Macro- scopic and microscopic quantification of enkephalin and substance P gene expression was carried out using computer-assisted image analysis. Tissue was collected from six control cases with no sign of neurological disease and six Huntington's disease cases ranging from grades 0 to 3 as determined by neuropathological evaluation. The clinical and pathological diagnosis of Huntington's disease was confirmed unequivocally by genetic analysis of the CAG repeat length in both copies of IT15, the Huntington's disease gene. A marked reduction in both enkephalin and substance P messenger RNAs was detected in all regions of the caudate nucleus and putamen in Huntington's disease grades 2/3 when compared to controls; in the dorsal caudate few enkephalin or substance P messenger RNA-positive cells were detected. For the early grade (0/1) Huntington's disease cases, a heterogeneous reduction in both enkephalin and substance P messenger RNAs was noted; for enkephalin messenger RNA the striatal autoradiograms displayed a conspicuous patchy appearance. Detailed cellular analysis of the dorsal caudate revealed a striking reduction in the number of enkephalin and substance P messenger RNA-positive cells detected and in the intensity of hybridization signal/cell. These data suggest that both the "indirect" GABA/enkephalin and "direct" GABA/substance P pathways are perturbed very early in the course of the disease and that these early changes in chemical signalling may possibly underlie the onset of clinical symptoms. Key words: basal ganglia, striatal compartments, in situ hybridization, Huntington's disease, neuropeptide gene expression. Expansion of a trinucleotide (CAG) glutamine repeat sequence in excess of 40 repeats within one copy of the human IT15 gene is now recognized as being the genetic basis of Huntington's disease (HD), 29 a pro- gressive neurological disorder characterized clinically by cognitive impairment, choreiform movements and akathesia. 34 How expansion of this polyglutamine sequence, located within the coding region of the gene, results in this neurological disorder is still unknown. Pathologically, HD is characterized by a loss of striatal medium-sized spiny GABA projection neurons; the earliest signs of cell loss appear in the dorsomedial caudate and dorsal putamen. 49 These striatal GABA projection cells comprise at least two tTo whom correspondence should be addressed. Abbreviations: ENK, enkephalin; GFAP, glial fibrillary acidic protein; GPe, globus pallidus externus; GPi, globus pallidus internus; HD, Huntington's disease; NADPH, reduced nicotinamide adenine dinucleotide phosphate; SP, substance P. chemical phenotypes: one phenotype expresses the neuropeptide enkephalin (ENK) and the other the neuropeptides substance P (SP) and dynorphin. TM In rodents, combined retrograde tracing and in situ hybridization studies have shown that each of these two neuropeptide systems predominantly innervates different target nuclei: GABA/ENK cells innervate the external pallidal segment (GPe), whilst GABA/SP cells innervate the entopeduncular nucleus (rat hom- ologue of the primate internal pallidal segment, GPi) and the substantia nigra. 18 In non-human primates, a similar topography of target innervation has been established, ~5,46 suggesting that the underlying chemical circuitry of the primate basal ganglia may be similar, although more complex. Initial biochemical 7,12 and immunocytochemical studies of advanced HD cases found a marked re- duction in GABA in the striatum 47 and in ENK- and SP-like immunoreactivity ~6in the GPe and substantia nigra (respectively), providing strong evidence for 1023