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The effect of in utero ethanol exposure on hippocampal mossy fibers: an HRP study
Developmental Brain Research, 15 (1984) 275-279 Elsevier
275
BRD 60027
Short Communications
The effect of in utero ethanol exposure on hippocampal mossy fibers: an HRP study JAMES R. WEST and DWIGHT R. PIERCE Department of Anatomy, College of Medicine, Universityof lowa, Iowa City, 1.4 52242 (U.S.A.) (Accepted April 17th, 1984) Key words: ethanol - - brain development - - teratology - - hippocampus - - mossy fibers - - horseradish peroxidase - - rats
Adult rats, exposed to ethanol in utero exhibit aberrant mossy fiber-like Timm staining in the distal infrapyramidal region of the hippocampus at midtemporal levels. The present study utilized the anterograde transport of HRP to verify that the aberrant pattern of Timm staining represented a terminal field of the dentate granule cells. One or more HRP-labeled mossy fiber bundles were shown to cross to the infrapyramidal side of the pyramidal cell layer primarily, but not exclusively, at the same septotemporal level where the aberrant terminal field was located. Within the past decade clinicians have defined a characteristic group of anomalies in some of the offspring from mothers who abused ethanol during their pregnanciesS. Now well documented, the fetal alcohol syndrome (FAS) consists of a group of specific facial characteristics, pre- and postnatal growth deficiency, microcephaly and varying degrees of mental retardation ]8. A few instances of severe central nervous system neuropathology have been reported in humansS,13. In an attempt to understand FAS, we have established a rat model to study the consequences of ethanol exposure on brain development6,22,23,25. Previously, we demonstrated that in utero exposure to a liquid diet containing 35% ethanol derived calories during days 1-21 of gestation produced permanent alterations in hippocampal mossy fiber topographyZ3,2~. Dense mossy fiber-like Timm staining was present in a distal (CA3a) infrapyramidal position at midtemporal hippocampal levels in most of those animals. While a few mossy fiber terminals are located there in normal animals 20, heavy distal infrapyramidal Timm staining is normally present in CA3a only in the rostral one-third of the hippocam-
pus 19,24. This suggests that the ethanol exposure induced a hypertrophied mossy fiber terminal field23. Several important questions concerning the aberrant (presumably mossy fiber) staining could not be answered with the passive Timm stain. First of all, does the aberrant staining actually represent a terminal field of the granule cell axons? A second related question concerns the pathway by which the aberrant terminal field is reached. Do the axons cross through the pyramidal cell layer to the infrapyramidal (stratum oriens) side at the same septotemporal level where the aberrant terminal field is found, or do they descend ventrally (as longitudinal mossy fibers) from the normally occurring distal infrapyramidal mossy fibers at more rostral hippocampal levels? The purpose of this paper was to investigate these questions by using the anterograde transport of horseradish peroxidase. Offspring from Sprague-Dawley rats (King Animal Laboratories, Oregon, WI) exposed to a liquid diet (Bio-Serv, PR-11) containing 35% ethanol derived calories during days 1-21 of gestation were cross-fostered at birth to normal dams and weaned at 22 days 23. The dams consumed from 10-12 g/kg of
Correspondence: J. R. West, Department of Anatomy, College of Medicine, University of Iowa, Iowa City, IA 52242, U.S.A. 0165-3806/84/$03.00 © 1984 Elsevier Science Publishers B.V.
276 ethanol each day, which was similar to that of previous studies6,22,23,25. A f t e r reaching an age of at least 90 days, rats of either sex were r a n d o m l y selected for the H R P experiments. Normal adult rats of a comparable age were used as controls. A previous study indicated that reduced caloric intake actually affected the distribution of the distal infrapyramidal mossy fiber terminal field in an opposite m a n n e r from that in the ethanol-exposed group (i.e. the reduced caloric input diminished rather than e x p a n d e d the septotemporal extent of the distal infrapyramidal mossy fiber terminal field) 23. Therefore, pair-fed controls were not examined in this study. O u r m e t h o d for the a n t e r o g r a d e transport of horseradish peroxidase was used 21. Small iontophoretic injections (2 u A for 90-120 s, through glass micropipettes with a tip d i a m e t e r of 2 0 - 5 0 # m ) of 20% (w/v) horseradish peroxidase ( H R P ) dissolved in 2% (v/v) dimethylsulfoxide ( D M S O ) were m a d e into the dentate gyrus ventral to the occipital bend of the hippocampus. Following survival periods of 16-24 h, the animals were perfused and their brains were removed and serial horizontal sections (parallel to the dorsal surface of the hemispheres) were cut at 40 ~ m using a freezing microtome. The free-floating sections were processed for H R P localization using te-
tramethyl benzidine 14. The tissue was m o u n t e d on chrome-alum subbed glass slides and allowed to dry. The tissue was then counterstained with thionmJ, and examined under both light- and dark-field illumination. The iontophoresis of such small volumes of H R P was not without some difficulties. Often no H R P was released using the given current and temporal parameters, yet slightly higher current for a longer period p r o d u c e d injection sites that were unacceptably large for this study. Therefore, bilateral injections were usually made. In many of those cases H R P labeling occurred on one side only. In cases where H R P was visible in both dentate gyri, no differences could be attributed to the double injections. Except as noted below, each of the 12 e t h a n o l - e x p o s e d and 20 normal animals r e p o r t e d in this study had a successful H R P injection into at least one of the dentate gyri at a m i d t e m p o r a l h i p p o c a m p a l level. The pattern of H R P - l a b e l e d mossy fibers observed in the normal rats was similar to that published previously 2,3,1Z2°,2~,26. A large suprapyramidal and a small proximal infrapyramidal bundle were labeled in each case (Fig. 1). The s u p r a p y r a m i d a l mossy fiber bundle could be followed in h i p p o c a m p a l field CA3 almost to the b o r d e r of CA1 (Fig. 1). The mos-
Fig. 1. HRP-labeled mossy fibers at a midtemporal hippocampal level from a normal adult rat. A: low power dark-field photomicrograph illustrating the suprapyramidal mossy fiber terminal band and an HRP injection site in the infrapyramidal blade of the dentate gyrus. Magnification bar = 500 pro. B: higher magnification of the CA3a (fimbrial) portion of the hippocampal section shown in A. The arrows point to the few normal HRP-labeled distal infrapyramidal mossy fibers. Magnification bar = 200/~m. The following abbreviations are used for this and subsequent figures: G, granule cell layer; H, hilus; IP, infrapyramidal; P, pyramidal cell layer; SP suprapyramidal; a, b, c, subfields of hippocampal field CA3; arrowhead indicates border between CA3 and CA1.
277 sy fiber tip turned ventrally for distances of 800-1200 /.tmTM. In agreement with our earlier findings, single mossy fiber axons, with their characteristic periodic swellings, could be seen crossing to the infrapyramidal side of the pyramidal cell layer or coursing for a short distance in that region (Fig. 1B). Occasionally, small bundles of two or three labeled axons were observed leaving the suprapyramidal bundle 2°. However, in normal rats, labeled bundles were never observed crossing to the infrapyramidal side in CA3a from the longitudinal (ventrally coursing) portion of the suprapyramidal bundle. Injections placed in more dorsorostral levels of the dentate gyrus in three instances revealed a normal distal infrapyramidal labeled bundle in each case 24. Ten of the twelve rats exposed to ethanol in utero, exhibited thick fingers of labeling leaving the suprapyramidal bundle to reach a distal infrapyramidal position at a midtemporal hippocampal level. The distal infrapyramidal terminal field was similar in size to the aberrant terminal field observed with the passive Timm histochemical stain23,25 (Fig. 2). The densest and most extensive distal infrapyramidal H R P labeling occurred at, and slightly ventral to, the septotemporal level of the injection site, which is consistent with previous reports of a slight temporal bend in the mossY fiber projection7, 20. In one other case, the dis-
tal infrapyramidal H R P labeling was restricted to the lateral portion of hippocampal subfield CA3a. Also in contrast to the normal cases, two instances of significant distal infrapyramidal labeling were observed ventral to the main suprapyramidal mossy fiber projection (Fig. 3). There was no evidence that the distal infrapyramidal terminal band was formed by mossy fibers coursing directly (in stratum oriens) from the proximal infrapyramidal terminal field in CA3c. Neither was there any evidence that the aberrant mossy fiber terminal band was formed by a contribution from longitudinally oriented distal infrapyramidal mossy fibers from their normally occurring more dorsorostral position 20. This report confirms and extends recent findings that prenatal exposure to ethanol throughout gestation produces a greatly hypertrophied distal infrapyramidal mossy fiber terminal band in hippocampal subfield CA3a at a midtemporal hippocampal level. In addition, it demonstrates the pathway by which the mossy fibers course to reach the aberrantly developed terminal field. The granule cell axons destined to reach the distal infrapyramidal terminal field continued with the other suprapyramidal mossy fibers (roughly transverse to the longitudinal hippocampal axis) and crossed the pyramidal cell layer primarily at the same septotemporal level where the aberrant
<
%
Fig. 2. HRP-labeled mossy fiber terminal fields in CA3a of a rat exposed to ethanol in utero. A: low power dark-field photomicrograph illustrating the mossy fiber terminal field and injection site. Magnification bar = 500 am. The injection was matched for location, and size with the case in Fig. 1A: B: higher magnification of the CA3a (fimbrial) portion of the hippocampal section shown in A. Arrows indicate aberrant distal infrapyramidal mossy fiber terminal field. Magnification bar = 200/~m. Arrows point to the aberrant HRP-labeled distal infrapyramidal mossy fiber terminal band.
278
Fig. 3. HRP-labeled distal mossy fiber terminal fields at a midtemporal hippocampal level in an adult rat following prenatal exposure to ethanol. This section contains temporally directed mossy fibers 5 sections (200/~m) ventral to the transversely oriented suprapyramidal labeling in CA3a-c. Arrows denote aberrantly positioned mossy fibers; dark-field illumination. Magnification bar = 200.,m.
mossy fiber terminal field was located. A b e r r a n t distal infrapyramidal mossy fibers in subfield CA3a have been induced by 4 different conditions4,9.10,es. The functional consequences of aberrant mossy fibers have not yet been determined. However, the a m o u n t of p r o x i m a l infrapyramidal mossy fibers has been correlated with poor two-way avoidance performance 16,17. A d u l t rats exposed in utero to ethanol also exhibit learning deficits, particularly a lack of response inhibition11.15. It is possible
1 Adams, J. C., Stabilizing and rapid thionin staining of TMB-based HRP reaction product, Neurosci. Lett., 17 (1980) 7-9. 2 Blackstad, T. W., Brink, K., Hem, J. and Jeune, B., Distribution of hippocampal mossy fibers in the rat: an experimental study with silver impregnation methods, J. comp. NeuroL, 138 (1970) 433-449. 3 Cajal, S., Ram6n y, Histologie du System Nerveux de l'Homme et des Vertdbr~s, Vol. H, L. Azoulay (trans), Maloine, Paris, 1911. 4 Cheema, S. S. and Lauder, J. M.. Infrapyramidal mossy fi-
that ethanol-induced dysmorphology such as the hypertrophied mossy fiber terminal field observed in this study is responsible for some of the central nervous system dysfunction observed in children with FAS. This research was supported in part by Grants AA03884 and AA05523 from the National Institute on Alcohol A b u s e and Alcoholism to J.R.W.
bers in the hippocampus of methylazoxymethanol acetateinduced microcephalic rats, Develop. Brain Res., 9 (1983) 411-415. 5 Clarren, S. K., Alvord, Jr., E. C., Sumi, S. M., Streissguth, A. P. and Smith, D. W., Brain malformations related to prenatal exposure to ethanol, J. Pediatr., 92 (1978) 64-67. 6 Dewey, S. L. and West, J. R., Evidence for altered lesioninduced sprouting in the dentate gyrus of adult rats exposed to ethanol in utero, Alcohol, 1 (1984) 81-88. 7 Gaarskjaer, F. B., Organization of the mossy fiber system of the rat studied in extended hippocampi. II. Experimental
279 analysis of fiber distribution with silver impregnation methods, J. comp. Neurol., 178 (1978) 73-88. 8 Jones, K. L., Smith, D. W., Uileland, C. N. and Streissguth, A. P., Pattern of malformation in offspring of chronic alcoholic mothers, Lancet, 1 (1973) 1267-1276. 9 Lauder, J. M. and Mugnaini, E., Infrapyramidal mossy fibers in the hippocampus of the hyperthyroid rat, Develop. Neurosci., 3 (1980) 248-265. 10 Laurberg, S. and Zimmer, J., Lesion-induced rerouting of hippocampal mossy fibers in developing but not in adult rats, J. comp. Neurol., 190 (1980) 627-650. 11 Lochry, E. A. and Riley, E. P., Retention of passive avoidance and T-maze escape in rats exposed to alcohol prenatally, Neurobehav. Toxicol., 2 (1980) 107-115. 12 Lorente de N6, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the ammonic system, J. Psychol. Neurol., 46 (1934) 113-177. 13 Peiffer, J., Majewski, F., Fishbach, H., Bierich, J. and Volk, B., Alcohol embryo and fetopathy: neuropathology of three children and three fetuses, J. Neurol. Sci., 41 (1979) 125-137. 14 Mesulam, M.-M., Tetramethylbenzidine for horseradish peroxidase histochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 15 Riley, E. P., Lochry, E. A. and Shapiro, N. R., Lack of response inhibition in rats prenatally exposed to ethanol, Psychopharmacology, 62 (1979) 47-52. 16 Schwegler, H. and Lipp, H.-P., Is there a correlation between hippocampal mossy fiber distribution and two-way avoidance performance in mice and rats? Neurosci. Lett., 23 (1981) 25-30. 17 Schwegler, H., Lipp, H.-P., Vander Loos, H. and Busel-
maier, H., Individual hippocampal mossy fiber distribution in mice correlates with two-way avoidance performance, Science, 214 (1981) 817-819. 18 Streissguth, A. P., Landesman-Dwyer, S., Martin, J. C. and Smith, D. W., Teratogenic effects of alcohol in humans and laboratory animals, Science, 209 (1980) 353-361. 19 Swanson, L. W., Wyss, J. M. and Cowan, W. M., An autoradiographic study of the organization of the intrahippocampal association pathways in the rat, J. comp. Neurol., 181 (1978) 681-716. 20 West, J. R., Distal infrapyramidal and longitudinal mossy fibers at a midtemporal hippocampal level, Brain Res. Bull., 10 (1983) 137-146. 21 West, J. R. and Black, A. C., Jr., Enhancing the anterograde movement of HRP to label sparse neuronal projections, Neurosci. Lett., 12 (1979) 35-40. 22 West, J. R., Dewey, S. L. and Cassell, M. D., Prenatal ethanol exposure alters the post-lesion reorganization (sprouting) of acetylcholinesterase staining in the dentate gyrus of the adult rat, Develop. Brain Res., 12 (1984) 83-95. 23 West, J. R. and Hodges-Savola, C. A., Permanent hippocampal mossy fiber hyperdevelopment following prenatal ethanol exposure, Neurobehav. Toxicol. Teratol., 5 (1983) 139-150. 24 West, J. R., Hodges, C. A. and Black, Jr., A. C., Distal infrapyramidal granule cell axons possess typical mossy fiber morphology, Brain Res. Bull., 6 (1981) 119-124. 25 West, J.R., Hodges, C. A. and Black, Jr., A. C., Prenatal ethanol exposure alters the organization of hippocampal mossy fibers, Science, 211 (1981)957-959. 26 West, J. R., Van Hoesen, G. W. and Kosel, K. C., A demonstration of hippocampal mossy fiber axon morphology using the anterograde transport of horseradish peroxidase, Exp. Brain Res., 48 (1982) 209-216.
275
BRD 60027
Short Communications
The effect of in utero ethanol exposure on hippocampal mossy fibers: an HRP study JAMES R. WEST and DWIGHT R. PIERCE Department of Anatomy, College of Medicine, Universityof lowa, Iowa City, 1.4 52242 (U.S.A.) (Accepted April 17th, 1984) Key words: ethanol - - brain development - - teratology - - hippocampus - - mossy fibers - - horseradish peroxidase - - rats
Adult rats, exposed to ethanol in utero exhibit aberrant mossy fiber-like Timm staining in the distal infrapyramidal region of the hippocampus at midtemporal levels. The present study utilized the anterograde transport of HRP to verify that the aberrant pattern of Timm staining represented a terminal field of the dentate granule cells. One or more HRP-labeled mossy fiber bundles were shown to cross to the infrapyramidal side of the pyramidal cell layer primarily, but not exclusively, at the same septotemporal level where the aberrant terminal field was located. Within the past decade clinicians have defined a characteristic group of anomalies in some of the offspring from mothers who abused ethanol during their pregnanciesS. Now well documented, the fetal alcohol syndrome (FAS) consists of a group of specific facial characteristics, pre- and postnatal growth deficiency, microcephaly and varying degrees of mental retardation ]8. A few instances of severe central nervous system neuropathology have been reported in humansS,13. In an attempt to understand FAS, we have established a rat model to study the consequences of ethanol exposure on brain development6,22,23,25. Previously, we demonstrated that in utero exposure to a liquid diet containing 35% ethanol derived calories during days 1-21 of gestation produced permanent alterations in hippocampal mossy fiber topographyZ3,2~. Dense mossy fiber-like Timm staining was present in a distal (CA3a) infrapyramidal position at midtemporal hippocampal levels in most of those animals. While a few mossy fiber terminals are located there in normal animals 20, heavy distal infrapyramidal Timm staining is normally present in CA3a only in the rostral one-third of the hippocam-
pus 19,24. This suggests that the ethanol exposure induced a hypertrophied mossy fiber terminal field23. Several important questions concerning the aberrant (presumably mossy fiber) staining could not be answered with the passive Timm stain. First of all, does the aberrant staining actually represent a terminal field of the granule cell axons? A second related question concerns the pathway by which the aberrant terminal field is reached. Do the axons cross through the pyramidal cell layer to the infrapyramidal (stratum oriens) side at the same septotemporal level where the aberrant terminal field is found, or do they descend ventrally (as longitudinal mossy fibers) from the normally occurring distal infrapyramidal mossy fibers at more rostral hippocampal levels? The purpose of this paper was to investigate these questions by using the anterograde transport of horseradish peroxidase. Offspring from Sprague-Dawley rats (King Animal Laboratories, Oregon, WI) exposed to a liquid diet (Bio-Serv, PR-11) containing 35% ethanol derived calories during days 1-21 of gestation were cross-fostered at birth to normal dams and weaned at 22 days 23. The dams consumed from 10-12 g/kg of
Correspondence: J. R. West, Department of Anatomy, College of Medicine, University of Iowa, Iowa City, IA 52242, U.S.A. 0165-3806/84/$03.00 © 1984 Elsevier Science Publishers B.V.
276 ethanol each day, which was similar to that of previous studies6,22,23,25. A f t e r reaching an age of at least 90 days, rats of either sex were r a n d o m l y selected for the H R P experiments. Normal adult rats of a comparable age were used as controls. A previous study indicated that reduced caloric intake actually affected the distribution of the distal infrapyramidal mossy fiber terminal field in an opposite m a n n e r from that in the ethanol-exposed group (i.e. the reduced caloric input diminished rather than e x p a n d e d the septotemporal extent of the distal infrapyramidal mossy fiber terminal field) 23. Therefore, pair-fed controls were not examined in this study. O u r m e t h o d for the a n t e r o g r a d e transport of horseradish peroxidase was used 21. Small iontophoretic injections (2 u A for 90-120 s, through glass micropipettes with a tip d i a m e t e r of 2 0 - 5 0 # m ) of 20% (w/v) horseradish peroxidase ( H R P ) dissolved in 2% (v/v) dimethylsulfoxide ( D M S O ) were m a d e into the dentate gyrus ventral to the occipital bend of the hippocampus. Following survival periods of 16-24 h, the animals were perfused and their brains were removed and serial horizontal sections (parallel to the dorsal surface of the hemispheres) were cut at 40 ~ m using a freezing microtome. The free-floating sections were processed for H R P localization using te-
tramethyl benzidine 14. The tissue was m o u n t e d on chrome-alum subbed glass slides and allowed to dry. The tissue was then counterstained with thionmJ, and examined under both light- and dark-field illumination. The iontophoresis of such small volumes of H R P was not without some difficulties. Often no H R P was released using the given current and temporal parameters, yet slightly higher current for a longer period p r o d u c e d injection sites that were unacceptably large for this study. Therefore, bilateral injections were usually made. In many of those cases H R P labeling occurred on one side only. In cases where H R P was visible in both dentate gyri, no differences could be attributed to the double injections. Except as noted below, each of the 12 e t h a n o l - e x p o s e d and 20 normal animals r e p o r t e d in this study had a successful H R P injection into at least one of the dentate gyri at a m i d t e m p o r a l h i p p o c a m p a l level. The pattern of H R P - l a b e l e d mossy fibers observed in the normal rats was similar to that published previously 2,3,1Z2°,2~,26. A large suprapyramidal and a small proximal infrapyramidal bundle were labeled in each case (Fig. 1). The s u p r a p y r a m i d a l mossy fiber bundle could be followed in h i p p o c a m p a l field CA3 almost to the b o r d e r of CA1 (Fig. 1). The mos-
Fig. 1. HRP-labeled mossy fibers at a midtemporal hippocampal level from a normal adult rat. A: low power dark-field photomicrograph illustrating the suprapyramidal mossy fiber terminal band and an HRP injection site in the infrapyramidal blade of the dentate gyrus. Magnification bar = 500 pro. B: higher magnification of the CA3a (fimbrial) portion of the hippocampal section shown in A. The arrows point to the few normal HRP-labeled distal infrapyramidal mossy fibers. Magnification bar = 200/~m. The following abbreviations are used for this and subsequent figures: G, granule cell layer; H, hilus; IP, infrapyramidal; P, pyramidal cell layer; SP suprapyramidal; a, b, c, subfields of hippocampal field CA3; arrowhead indicates border between CA3 and CA1.
277 sy fiber tip turned ventrally for distances of 800-1200 /.tmTM. In agreement with our earlier findings, single mossy fiber axons, with their characteristic periodic swellings, could be seen crossing to the infrapyramidal side of the pyramidal cell layer or coursing for a short distance in that region (Fig. 1B). Occasionally, small bundles of two or three labeled axons were observed leaving the suprapyramidal bundle 2°. However, in normal rats, labeled bundles were never observed crossing to the infrapyramidal side in CA3a from the longitudinal (ventrally coursing) portion of the suprapyramidal bundle. Injections placed in more dorsorostral levels of the dentate gyrus in three instances revealed a normal distal infrapyramidal labeled bundle in each case 24. Ten of the twelve rats exposed to ethanol in utero, exhibited thick fingers of labeling leaving the suprapyramidal bundle to reach a distal infrapyramidal position at a midtemporal hippocampal level. The distal infrapyramidal terminal field was similar in size to the aberrant terminal field observed with the passive Timm histochemical stain23,25 (Fig. 2). The densest and most extensive distal infrapyramidal H R P labeling occurred at, and slightly ventral to, the septotemporal level of the injection site, which is consistent with previous reports of a slight temporal bend in the mossY fiber projection7, 20. In one other case, the dis-
tal infrapyramidal H R P labeling was restricted to the lateral portion of hippocampal subfield CA3a. Also in contrast to the normal cases, two instances of significant distal infrapyramidal labeling were observed ventral to the main suprapyramidal mossy fiber projection (Fig. 3). There was no evidence that the distal infrapyramidal terminal band was formed by mossy fibers coursing directly (in stratum oriens) from the proximal infrapyramidal terminal field in CA3c. Neither was there any evidence that the aberrant mossy fiber terminal band was formed by a contribution from longitudinally oriented distal infrapyramidal mossy fibers from their normally occurring more dorsorostral position 20. This report confirms and extends recent findings that prenatal exposure to ethanol throughout gestation produces a greatly hypertrophied distal infrapyramidal mossy fiber terminal band in hippocampal subfield CA3a at a midtemporal hippocampal level. In addition, it demonstrates the pathway by which the mossy fibers course to reach the aberrantly developed terminal field. The granule cell axons destined to reach the distal infrapyramidal terminal field continued with the other suprapyramidal mossy fibers (roughly transverse to the longitudinal hippocampal axis) and crossed the pyramidal cell layer primarily at the same septotemporal level where the aberrant
<
%
Fig. 2. HRP-labeled mossy fiber terminal fields in CA3a of a rat exposed to ethanol in utero. A: low power dark-field photomicrograph illustrating the mossy fiber terminal field and injection site. Magnification bar = 500 am. The injection was matched for location, and size with the case in Fig. 1A: B: higher magnification of the CA3a (fimbrial) portion of the hippocampal section shown in A. Arrows indicate aberrant distal infrapyramidal mossy fiber terminal field. Magnification bar = 200/~m. Arrows point to the aberrant HRP-labeled distal infrapyramidal mossy fiber terminal band.
278
Fig. 3. HRP-labeled distal mossy fiber terminal fields at a midtemporal hippocampal level in an adult rat following prenatal exposure to ethanol. This section contains temporally directed mossy fibers 5 sections (200/~m) ventral to the transversely oriented suprapyramidal labeling in CA3a-c. Arrows denote aberrantly positioned mossy fibers; dark-field illumination. Magnification bar = 200.,m.
mossy fiber terminal field was located. A b e r r a n t distal infrapyramidal mossy fibers in subfield CA3a have been induced by 4 different conditions4,9.10,es. The functional consequences of aberrant mossy fibers have not yet been determined. However, the a m o u n t of p r o x i m a l infrapyramidal mossy fibers has been correlated with poor two-way avoidance performance 16,17. A d u l t rats exposed in utero to ethanol also exhibit learning deficits, particularly a lack of response inhibition11.15. It is possible
1 Adams, J. C., Stabilizing and rapid thionin staining of TMB-based HRP reaction product, Neurosci. Lett., 17 (1980) 7-9. 2 Blackstad, T. W., Brink, K., Hem, J. and Jeune, B., Distribution of hippocampal mossy fibers in the rat: an experimental study with silver impregnation methods, J. comp. NeuroL, 138 (1970) 433-449. 3 Cajal, S., Ram6n y, Histologie du System Nerveux de l'Homme et des Vertdbr~s, Vol. H, L. Azoulay (trans), Maloine, Paris, 1911. 4 Cheema, S. S. and Lauder, J. M.. Infrapyramidal mossy fi-
that ethanol-induced dysmorphology such as the hypertrophied mossy fiber terminal field observed in this study is responsible for some of the central nervous system dysfunction observed in children with FAS. This research was supported in part by Grants AA03884 and AA05523 from the National Institute on Alcohol A b u s e and Alcoholism to J.R.W.
bers in the hippocampus of methylazoxymethanol acetateinduced microcephalic rats, Develop. Brain Res., 9 (1983) 411-415. 5 Clarren, S. K., Alvord, Jr., E. C., Sumi, S. M., Streissguth, A. P. and Smith, D. W., Brain malformations related to prenatal exposure to ethanol, J. Pediatr., 92 (1978) 64-67. 6 Dewey, S. L. and West, J. R., Evidence for altered lesioninduced sprouting in the dentate gyrus of adult rats exposed to ethanol in utero, Alcohol, 1 (1984) 81-88. 7 Gaarskjaer, F. B., Organization of the mossy fiber system of the rat studied in extended hippocampi. II. Experimental
279 analysis of fiber distribution with silver impregnation methods, J. comp. Neurol., 178 (1978) 73-88. 8 Jones, K. L., Smith, D. W., Uileland, C. N. and Streissguth, A. P., Pattern of malformation in offspring of chronic alcoholic mothers, Lancet, 1 (1973) 1267-1276. 9 Lauder, J. M. and Mugnaini, E., Infrapyramidal mossy fibers in the hippocampus of the hyperthyroid rat, Develop. Neurosci., 3 (1980) 248-265. 10 Laurberg, S. and Zimmer, J., Lesion-induced rerouting of hippocampal mossy fibers in developing but not in adult rats, J. comp. Neurol., 190 (1980) 627-650. 11 Lochry, E. A. and Riley, E. P., Retention of passive avoidance and T-maze escape in rats exposed to alcohol prenatally, Neurobehav. Toxicol., 2 (1980) 107-115. 12 Lorente de N6, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the ammonic system, J. Psychol. Neurol., 46 (1934) 113-177. 13 Peiffer, J., Majewski, F., Fishbach, H., Bierich, J. and Volk, B., Alcohol embryo and fetopathy: neuropathology of three children and three fetuses, J. Neurol. Sci., 41 (1979) 125-137. 14 Mesulam, M.-M., Tetramethylbenzidine for horseradish peroxidase histochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 15 Riley, E. P., Lochry, E. A. and Shapiro, N. R., Lack of response inhibition in rats prenatally exposed to ethanol, Psychopharmacology, 62 (1979) 47-52. 16 Schwegler, H. and Lipp, H.-P., Is there a correlation between hippocampal mossy fiber distribution and two-way avoidance performance in mice and rats? Neurosci. Lett., 23 (1981) 25-30. 17 Schwegler, H., Lipp, H.-P., Vander Loos, H. and Busel-
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