EVIDENCE FOR A DEFICIT IN CHOLINERGIC INTERNEURONS IN THE STRIATUM IN SCHIZOPHRENIA D. J. HOLT,*² M. M. HERMAN,‡ T. M. HYDE,‡ J. E. KLEINMAN,‡ C. M. SINTON,§ D. C. GERMAN,§ L. B. HERSH, k A. M. GRAYBIEL¶ and C. B. SAPER* ** *Department of Neurology, Beth Israel Deaconess Medical Center and the Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, U.S.A. ²The Committee on Neurobiology, The University of Chicago, Chicago, Illinois, U.S.A. ‡Clinical Brain Disorders Branch, NIMH, IRP, NIH, Bethesda, Maryland, U.S.A. §Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A. k Department of Biochemistry, University of Kentucky, Lexington, Kentucky, U.S.A. ¶Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Boston, Massachusetts, U.S.A. Abstract —Neurochemical and functional abnormalities of the striatum have been reported in schizophrenic brains, but the cellular substrates of these changes are not known. We hypothesized that schizophrenia may involve an abnormality in one of the key modulators of striatal output, the cholinergic interneuron. We measured the densities of cholinergic neurons in the striatum in schizophrenic and control brains in a blind analysis, using as a marker of this cell population immunoreactivity for choline acetyltransferase, the synthetic enzyme of acetylcholine. As an independent marker, we used immunoreactivity for calretinin, a protein which is co-localized with choline acetyltransferase in virtually all of the cholinergic interneurons of the striatum. A significant decrease in choline acetyltransferase-positive and calretinin-positive cell densities was found in the schizophrenic cases compared with controls in the striatum as a whole [for the choline acetyltransferase-positive cells: controls: 3.21 ^ 0.48 cells/mm 2 (mean ^ S.D.), schizophrenics: 2.43 ^ 0.68 cells/mm 2 ; P  0.02]. The decrease was patchy in nature and most prominent in the ventral striatum (for the choline acetyltransferase-positive cells: controls: 3.47 ^ 0.59 cells/mm 2 , schizophrenics: 2.52 ^ 0.64 cells/ mm 2 ; P  0.005) which included the ventral caudate nucleus and nucleus accumbens region. Three of the schizophrenic cases with the lowest densities of cholinergic neurons had not been treated with neuroleptics for periods from more than a month to more than 20 years. A decrease in the number or function of the cholinergic interneurons of the striatum may disrupt activity in the ventral striatal– pallidal–thalamic –prefrontal cortex pathway and thereby contribute to abnormalities in function of the prefrontal cortex in schizo- phrenia.  1999 IBRO. Published by Elsevier Science Ltd. Key words: schizophrenia, striatum, cholinergic, interneuron, human, basal ganglia. Several lines of evidence implicate the striatum in the pathophysiology of schizophrenia. Disorders that are known to affect the striatum such as Huntington’s disease and Wilson’s disease may produce psychiatric symptoms in addition to motor dysfunction; these symptoms can be diffi- cult to distinguish from those of schizophrenia. 1,27,60,74 Simi- larly, drugs used to treat hypokinetic movement disorders such as dopaminergic agonists or muscarinic cholinergic antagonists, which are presumed to act in the striatum, can precipitate psychotic symptoms, including hallucina- tions and delusions. 17,40,62 Abnormal movements, such as dyskinesias, are observed in medication-naive schizo- phrenics, 61 suggesting that a striatal abnormality could account for both the motor and psychiatric symptoms of schizophrenics. The striatum has been shown in some studies to have abnormally low metabolic activity in schizo- phrenics, which can be normalized after treatment with neuroleptic medication. 14,15,30,50,70,89 Studies have also shown increases in volume of portions of the striatum and globus pallidus of schizophrenics, which may be an effect of neuroleptic medication. 10,13,47,49,56 There have been recent reports of decreased 77 and increased cell number 5 in the striatum of schizophrenics and an ultrastructural study revealing a decrease in the size of dendritic spines in the striatum of schizophrenics. 84 A number of recent studies has reported abnormalities in the levels of various neuro- transmitter receptors in the striatum of schizophrenics, 4,44,71 including a report of a reduction in M1 muscarinic receptor levels. 28 However, few studies have examined the distribution of a specific striatal cell type in the brains of schizophrenic patients. 46 A deficit in cholinergic neurotransmission may be similar in its effects and potentially indistinguishable clinically from an excess of dopaminergic transmission in the striatum. 40,62 Consequently, the psychosis of schizophrenia, which is ameliorated by dopamine antagonists, 67 could also result at least in part from a deficit in cholinergic neurotransmission. One previous study reported a reduction in levels of choline acetyltransferase (ChAT), the synthetic enzyme for acetyl- choline, in the nucleus accumbens of schizophrenics, 6 but other studies have disputed this result. 7,25,32,68 Because cholin- ergic neurons are not homogeneously distributed in the stria- tum, 52–54 the discrepancies in previous assay studies may in part reflect differences in sampling techniques. To avoid this problem, in this study we have used immunocytochemistry for neurochemical markers of cholinergic striatal interneurons to assess whether there is a change in the density of this cell Cholinergic deficit in schizophrenic striatum 21 21 Neuroscience Vol. 94, No. 1, pp. 21–31, 1999 Copyright  1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 PII: S0306-4522(99)00279-1 Pergamon **To whom correspondence should be addressed. Abbreviations: ChAT, choline acetyltransferase; PBS, phosphate-buffered saline; PBS-H, PBS with 10% horse serum; PBS-M, PBS with 0.25% Triton X-100 and non-fat dry milk.