City University of New York (CUNY) CUNY Academic Works All Dissertations, Theses, and Capstone Projects Dissertations, Theses, and Capstone Projects 1989 Amino Acids and N-acetyl-aspartyl-glutamate as Neurotransmitter Candidates in the Monkey Retinogeniculate Pathways Ricardo A. Molinar-Rode Graduate Center, City University of New York How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/gc_etds/1641 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] INFORMATION TO USERS The most advanced technology has been used to photo­ graph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. 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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Com pany 300 North Z eeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9000716 Amino acids and N-acetyl-aspartyl-glutamate as neurotransmitter candidates in the monkey retinogeniculate pathways Molinar-Rode, Ricardo A., Ph.D. City University of New York, 1989 UMI 300 N. ZeebRd. Ann Arbor, MI 48106 ■lLL.jtjm m im ,Liii--LiiijL. 11 jiibii. pm.k.i ■ AMINO ACIDS AND N-ACETYL-ASPARTYL-GLUTAMATE AS NEUROTRANSMITTER CANDIDATES IN THE MONKEY RETINOGENICULATE PATHWAYS by Ricardo A. Molinar-Rode A dissertation submitted to the Graduate Faculty in Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The City University of New York. 1989 This manuscript has been read and accepted for the Graduate Faculty in Biomedical Sciences in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy. [signature] <2./* 3/ W Date ChairmarPof the Examining Committee Pedro Pasik, M.D. [signature] X f'lT.U'S Dat a ' Terry Krulwich, Ph.D. Bernard Cohen, M.D. Soil Berl, M.D. Andreas Plaitakis, M.D. Efrain Azmitia, Ph.D. Supervisory Committee The City University of New York ABSTRACT AMINO ACIDS AND N-ACETYL-ASPARTYL-GLUTAMATE AS NEUROTRANSMITTER CANDIDATES IN THE MONKEY RETINOGENICULATE PATHWAYS by Ricardo A. Molinar-Rode Advisor: Professor Pedro Pasik The lateral geniculate nucleus (LGNd) receives chemically identified inputs from brain stem structures, the thalamus and visual cortex. The identity of the neurotransmitter(s) in the retinal input, however, is unknown. To investigate the possibility that some amino acids and certain dipeptides, such as N-acetyl-aspartyl-glutamate (NAAG), fulfill this function, changes in their concentration were measured in the optic tract, parvocellular and magno- cellular segments of the LGNd, superior colliculus and visual cortex of six monkeys (Macaca fascicularis) , seven days after right optic tractotomy. The LGNd was studied also in two additional macaques, three months after occipital lobectomy. Tissue was frozen within five minutes of death, regions were dissected with the micropunch technique, and substances were analyzed by HPLC. Of the ten compounds measured in the normal side, gluta­ mate, glutamine, glycine, and alanine had homogeneous distri­ butions. GABA was highest in the superior colliculus, cysta­ thionine and NAAG decreased in the rostrocaudal direction, and N-acetyl-aspartate showed an opposite gradient of concentra­ tion. The heterogeneity in taurine and aspartate was less systematic. Optic tract section induced significant, large reductions in NAAG, glutamate and aspartate in the optic tract distal to the lesion. Significant decreases in NAAG, and to a lesser extent in glutamate, were observed in the LGNd. Changes in the dipeptide were apparent in both the parvocellular and magnocellular segments. Reductions in glutamate reached significance in the parvocellular laminae, and those of aspartate approached significance in the magnocellular division. No significant differences were detected in the superior colliculus and striate cortex. Occipital lobectomy produced large declines in aspartate and glutamate in the LGNd, as well as moderate reductions in alanine and GABA, and minor changes in glutamine and glycine. The results of optic tractotomy support the role of NAAG as a neurotransmitter candidate in the monkey retinogeniculate pathways; its significant decrease in both geniculate segments suggests that X- and Y- retinal axons utilize this substance. Although at times the reductions in glutamate or aspartate failed to reach significance, their role cannot be excluded. The findings after occipital lobectomy strongly favor these latter substances as corticogeniculate transmitters. ACKNOWLEDGEMENTS I wish to express my appreciation to: Dr. Pedro Pasik, my advisor, for his consistent guidance throughout the course of this project, and for his exceptional advice in writing this manuscript. The members of my supervisory committee, for their interest in my work and important suggestions. I thank especially Dr. Soli Berl, for his support and scientific counsel, and Dr. Andreas Plaitakis, for his advice in biochemical methods. I extend my gratitude to the members of the Neurology Department, in particular to Dr. Catherine Mytilineou, for her support at some difficult moments. Dr. Terry Krulwich, and the organizers of the Doctoral Program in Neurobiology, for giving me the opportunity to accomplish my education in this exciting field. Drs. Andrew Glover, Gay Holstein and Julian Martinez, for fruitful discussions, and for helping to maintain an enthu­ siastic, supportive working environment. Victor Rodriguez and Rosemary Lang, for their technical assistance, and expertise in photography. The members of the Clinical Trials Unit at Mount Sinai, for their friendship, assistance, and for introducing me to the use of WordPerfect and some graphics programs. My family, friends and especially Liliana, for their love and support. v TABLE OF CONTENTS Title page i Approval page ii Abstract iii Acknowledgements V Table of contents vi List of tables ix List of figures X I. INTRODUCTION 1 II. BACKGROUND 4 1. MORPHOLOGICAL FEATURES OF THE MONKEY DORSAL LATERAL GENICULATE NUCLEUS 4 1.1 NORMAL STRUCTURE 4 1.2 DEGENERATIVE CHANGES 14 2. NEUROACTIVE SUBSTANCES IN THE LATERAL GENICULATE NUCLEUS 21 2.1 AMINO ACIDS 21 A) GLUTAMATE AND ASPARTATE 21 B) GABA 38 C) GLYCINE 47 2.2 PEPTIDES 49 A) SUBSTANCE P 49 B) ENKEPHALIN 52 C) CHOLECYSTOKININ 53 D) SOMATOSTATIN 55 E) OTHERS 57 2.3 ACETYLCHOLINE 62 2.4 MONOAMINES 68 A) SEROTONIN 68 B) NORADRENALINE 72 C) ADRENALINE 76 vi D) DOPAMINE 77 III. MATERIALS AND METHODS 79 1. SUBJECTS 79 2. PROCEDURES 79 2.1 SURGERY 79 2.1.1 RIGHT OPTIC TRACT SECTION 79 2.1.2 LEFT OCCIPITAL LOBECTOMY 84 2.2 NEUROLOGICAL EXAMINATION 85 2.3 SPECIMEN COLLECTION 88 2.4 HISTOLOGY 90 2.5 TISSUE PREPARATION 90 2.6 BIOCHEMICAL ANALYSES 101 2.6.1 AMINO ACID ANALYSIS 101 2.6.2 NASP AND NAAG ANALYSIS 108 2.7 STATISTICAL ANALYSES 111 2.7.1 CONTROL (NORMAL) STRUCTURES 111 2.7.2 CONTROL AGAINST EXPERIMENTAL STRUCTURES 112 IV. RESULTS 114 1. NEUROLOGICAL EXAMINATION 114 1.1 RIGHT OPTIC TRACTOTOMY 114 1.2 LEFT OCCIPITAL LOBECTOMY 115 2. HISTOLOGICAL VERIFICATION 115 2.1 RIGHT OPTIC TRACTOTOMY 115 2.2 LEFT OCCIPITAL LOBECTOMY 116 3. BIOCHEMICAL ANALYSES 121 3.1 AMINO ACID ANALYSIS 121 3.1.1 CRITERIA FOR EXCLUSION/INCLUSION 121 3.1.2 NORMAL REGIONAL DISTRIBUTION 12 6 3.1.3 EFFECTS OF LESIONS 139 A) OPTIC TRACT SECTION 139 B) OCCIPITAL LOBECTOMY 151 vii 3.2 NAAG AND NASP ANALYSIS 153 3.2.1 NORMAL REGIONAL DISTRIBUTION 153 3.2.2 EFFECTS OF OPTIC TRACT SECTION 163 V. DISCUSSION 169 VI. REFERENCES 191 viii LIST OF TABLES Amino acid concentration in the visual system 23 Glutamate-related binding in the visual system 33 Substance-P concentration in the visual system 51 Experimental subjects 80 Amounts of tissue collected for analyses 98 HPLC gradient program for amino acid analysis 102 Fluorescence constants for some amino acids 123 Normal regional distribution of amino acids in the monkey visual system 130 Effect of optic tractotomy on the amino acid concentration in the monkey visual system 141 Effect of occipital lobectomy on the amino acid concentration in the lateral geniculate nucleus 152 Normal