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Labeling of human retinohypothalamic tract with the carbocyanine dye, DiI
Brain Research, 560 (1991) 297-302 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124813K
BRES 24813
297
Short Communications
Labeling of human retinohypothalamic tract with the carbocyanine dye, DiI Deborah I. Friedman 1'2, J. Kelly Johnson 4'*, Regina L. Chorsky 3 and Edward G. Stopa 3'5 Departments of 1Neurology, 2Ophthalmology and 3pathology [Division of Neuropathology], SUNY Health Science Center, Syracuse, NY 13210 (U.S.A.), 41nstitutefor Sensory Research, Syracuse University, Syracuse, NY 13210 (U.S.A.) and 5Brain Tissue Resource Center, McLean Hospital, Harvard Medical School, Belmont, MA 02178 (U.S.A.)
(Accepted 28 May 1991) Key words: Retinohypothalamic tract; Suprachiasmatic nucleus; Hypothalamus; Carbocyanine dye; DiI; Circadian rhythm
The carboeyanine dye, DiI, was used to demonstrate human retinohypothalamic tract (RHT) projections in 6 normal human postmortem brains. In 5 of 6 brains, labeling was seen extending from the site of implantation in the distal optic nerve to both the ipsilateral and contralateral suprachiasmatic nuclei. This study confirms the presence of RHT projections in humans, and demonstrates the usefulness of DiI for neuronal tracing in human postmortem tissue. The suprachiasmatic nucleus (SCN) in the anterior hypothalamus has been found to be important for synchronizing circadian rhythms to the environmental light-dark cycle 19'21'28'31. Early studies in rodents demonstrated that afferent visual pathways were necessary for relaying information regarding environmental illumination to the SCN. Bilateral transection of the optic nerve was found to produce desynchronization of circadian rhythmicity, whereas transection distal to the optic chiasm did not alter circadian regulation 16'22'23'31. These observations suggested the presence of a direct retinofugal pathway to the SCN that terminated near the optic chiasm, the retinohypothalamic tract (RHT). Subsequent anatomical studies in numerous species have confirmed that the SCN receives direct retinal projections by way of the R H T 2A3'2°'24'26. In addition, a second indirect retinal projection has been identified from the intergeniculate leaflet, the geniculohypothalamic tract 34. In humans, definitive demonstration of the R H T has n o t been feasible using standard neuroanatomical tracing techniques that generally require viable tissue. However, R H T projections to the human SCN and paraventricular nucleus have been described using the paraphenylene diamine (PPD) technique in patients with long-standing monocular optic nerve damage 29'3°. The utility of the PPD technique is derived from its ability to stain degenerated axon remnants that have persisted in postmortem tissue for long periods of time after the ini-
tial lesion has occurred. Therefore, PPD is a useful anatomical marker of afferent visual pathways when a discrete optic nerve lesion is present, but it cannot be used to examine neuronal pathways in normal brains. Carbocyanine dyes are lipophilic fluorescent substances that provide anterograde and retrograde staining of neurons in vivo and in vitro 35. When applied to brain tissue or peripheral nerve endings in vivo, the dyes are taken up by axons or axon terminals and actively transported to cell bodies 3'14. In fixed tissue, they become inserted into the lipid fraction of the plasma membrane and diffuse freely down the concentration gradient. Labeling has been demonstrated in aldehyde-fixed neuronal tissue in several species 9'1°'12A8'38. DiI (1,1"-dioctadecyl3,3,3",3"-tetramethylindocarbocyanine perchlorate; Molecular Probes, Eugene, OR) has proven to be useful for studying the visual system in various submammalian and mammalian species 4'9'10'15'35-37'39. In the present study we used DiI to confirm the presence of retinal projections to the SCN in normal human brains. Six adult human brains were obtained within 24 h of death. All brains were confirmed to be free of disease by both clinical history and postmortem neuropathological examination. A block of tissue containing the optic nerves, optic chiasm, and hypothalamus was dissected from the fresh brain, and immersed in 4% paraformaldehyde in 0.1 M Sorensen's phosphate buffer (pH 7.2). Some of the brains were briefly immersed in 10% for-
* Current address: Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-2715, U.S.A. Correspondence: E.G. Stopa, Department of Pathology, SUNY Health Science Center, 750 East Adams Street, Syracuse, NY 13210, U.S.A.
298
Fig. 1. Dil crystals implanted into the left optic nerve of a human brain, fixed in 4% paraformaldehyde.
malin after brain removal, and the specimens were placed in 4% p a r a f o r m a l d e h y d e following dissection. Within 24-48 h of death, DiI crystals were e m b e d d e d into the distal end of one optic nerve using an operating microscope, after excess solution had been blotted (Fig. 1). The tissue was then carefully placed back into 4% phosphate-buffered p a r a f o r m a l d e h y d e , and maintained at 37 °C in darkness. A f t e r approx. 3 - 4 months, the hypothalamus was sectioned coronally at 60-100/~m using a vibratome. The sections were collected on gelatin-coated slides, m o u n t e d in an aqueous mounting m e d i u m (Gelmount, B i o m e d a Corp., Foster City, C A ) , and photographed with a fluorescence microscope (Nikon Optiphot, O l y m p u s Vanox) using a standard r h o d a m i n e
filter set. To ensure the long-term stability of the DiI label, the slides were stored at 4 °C in darkness whenever they were not being viewed. In 2 additional h u m a n hypothalami, 50-/~m coronal sections through the SCN were processed for vasoactive intestinal polypeptide (VIP) immunocytochemistry and osmium stained in accordance with p r o c e d u r e s previously described 2. In 5 of the 6 brains, DiI labeling was found to diffuse from the site of implantation in the optic nerve through the optic chiasm, to both the ipsilateral and contralateral SCN (Figs. 2-4). These labeled axonal projections e n t e r e d the ventral surface of the SCN in a manner similar to that described in rodents 2°'24"26. L a b e l e d fibers were also seen decussating within the optic chiasm (Fig.
Fig. 2. Composite montage of DiI fluorescence within the optic chiasm and rostral preoptic hypothalamus, 4 months after the implantation of DiI into the left optic nerve (rhodamine filter). Note the projections to the ipsilateral (arrow) and contralateral suprachiasmatic nuclei (SCN). Some optic nerve fibers can be seen decussating within the chiasm. Fig. 3. Fluorescent fibers can be seen crossing beneath the third ventricle to the contralateral SCN. Fig. 4. High magnification view demonstrating numerous fluorescent fibers within the ipsilateral SCN (top half of picture), arising from the optic chiasm (lower half of picture). The third ventricle can be seen on the left edge of the photograph. Fig. 5. High magnification views of fluorescent fibers within the optic chiasm. Individual fibers can be readily traced as they course across the microscopic field.
v-
ii,,
~i ¸
301 5). Fibers leaving the chiasm en route to the ipsilateral SCN were generally well defined, and their orientation correlated with vertically oriented myelinated fibers observed in this region on osmium-stained preparations (Fig. 6A,B). Correlations between osmium/VIP stained sections and DiI preparations made at comparable anatomic levels confirmed that the DiI labeled fibers were entering the SCN. The SCN is the only site in the anterior hypothalamus that contains a VIP immunopositive neuronal subpopulation 33. Several factors were found to contribute to the overall effectiveness and utility of carbocyanine tracers in postmortem human brain. The rate of DiI labeling was enhanced when the specimens were stored at 37 °C rather than at r o o m temperature. Even under optimum conditions (37 °C with 4% paraformaldehyde fixative), we observed a diffusion rate of only 10 m m after 3 months. Whether or not premortem events also play a role in modulating diffusion rates remains to be determined. The DiI label was more robust in some brains than others, and in one brain it failed to diffuse. Although it is impossible to determine the reason for failure of DiI labeling in this brain with certainty, we speculate that the brief period of initial 10% formalin fixation may be an important contributing factor. Formalin-fixed tissue was generally found to be less suitable than tissue fixed in 4% paraformaldehyde, possibly due to the impurities present in many commercially obtained formalin preparations. After tissues were sectioned, mounted, and viewed, DiI began to fade within approx. 72 h of mounting unless the slides were stored at 4 °C in darkness. Disappearance of the DiI label has been described in association both for the avidin-biotin and peroxidase-antiperoxidase methods, as well as during the incubation of tissue in 0.04% hydroperoxide or 1% sodium borohydride 8.
O u r findings are in agreement with the previous PPD analysis by Sadun and coworkers 29, and confirm the presence of both ipsilateral and contralateral projections from the retina to the ventral human SCN. The improved visualization made possible by the DiI technique provides additional evidence that R H T pathways may be important in mediating various clinically observed phenomena 25. For example, abnormalities of the neuroendocrine regulatory system have been attributed to the absence of diurnal light-dark input in blind persons. Blind women experience earlier menarche than sighted women, and have differences in water, insulin, and carbohydrate balances compared to sighted individuals 11. Manipulation of the light-dark cycle has been shown to shift human circadian rhythms, and has been useful in the treatment of affective disorders 17'27. Additionally, it has been shown that light is a potent entraining stimulus for human circadian rhythms 7, and that exposure to bright light can reset the human circadian pacemaker independently of the sleep-wake cycles 5. Light exposure has also been shown to improve adaptation from daytime to nighttime shift work 6. In conclusion, this study provides further evidence for the existence of the R H T in normal human brains, and demonstrates the usefulness of DiI for neuronal tracing in human postmortem tissue. Future applications of the DiI technique may provide new insights into the anatomy and development of the human nervous system, which were not previously feasible using classical neuroanatomical tracing techniques.
1 Anthony, E.L.P. and King, J.C., Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system, Am. J. Anat., 175 (1986) 179-195. 2 Cassone, V.M., Speh, J.C., Card, J.P. and Moore, R.Y., Comparative anatomy of the mammalian hypothalamic suprachiasmatic nucleus, J. Biol. Rhythms, 3 (1988) 71-93. 3 Catsicas, S., Thanos, S. and Clarke, P.G.H., Initial imprecision in the topography of the chick embryo's isthmo-optic projection, Soc. Neurosci. Abstr., 12 (1986) 984. 4 Collelo, R.J. and Guillery, R.W., The early development of retinal ganglion cells with uncrossed axons in the mouse: retinal position and axonal course, Development, 108 (1990) 515523. 5 Czeisler, C.A., Allan, J.S., Strogatz, S.H., Ronda, J.M., Sanchez, R., Rios, C.D., Freitag, W.O., Richardson, G.S. and Kronauer, R.E., Bright light resets the human circadian pacemaker independently of the timing of the sleep-wake cycle, Science, 233 (1986) 667-671. 6 Czeisler, C.A., Johnson, M.P., Duffy, J.E, Brown, E.N.,
Ronda, J.M. and Kronauer, R.E., Exposure to bright light and darkness to treat physiologic maladaptation to night work, N. Engl. J. Med., 322 (1990) 1253-1259. Czeisler, C.A., Kronauer, R.E., Allan, J.A., Duffy, J.F., Jewett, M.E., Brown, E.N. and Ronda, J.M., Bright light induction of strong (Type O) resetting of the human circadian pacemaker, Science, 244 (1989) 1328-1333. Elberger, A.J. and Honig, M.G., Double-labeling of tissue containing the carbocyanine dye DiI for immunocytochemistry, J. Histochem. Cytochem., 38 (1990) 735-739. Godement, P., Salaun, J. and Mason, C.A., Retinal axon pathfinding in the optic chiasm: divergence of crossed and uncrossed fibers, Neuron, 5 (1990) 173-186. Godement, P., Vanselow, J., Thanos, S. and Bonhoeffer, E, A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue, Development, 101 (1987) 697-713. Hasan, W., Chandra, P. and Prasad, J.N., Continuous illumination and oogenesis in albino rabbits, Ind. J. Physiol. Pharmacol., 25 (1981) 279-284.
The authors wish to acknowledge Drs. Stephen Chamberlain, Andrew Satlin, George Collins and Ellion Albers for many helpful suggestions, and Dr. Edward Bird for providing some of the tissues used in this study. Supported by A ~ 9 5 and AG09301 (E.G.S.), Brain Bank Grant MI-I/NS31862, SUNY Health Science Center Pathology Medical Service Group (E.G.S.) and the Hendricks Fund for Medical Research (D.I.E, E.G.S.).
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302 12 Heffner, C.D., Lumsden, A.G.S. and O'Leary, D.D.M., Target control of collateral extension and directional axon growth in the mammalian brain, Science, 12 (1990) 217-220. 13 Herrick, C.J., The hypothalamus of Necturus, J. Comp. Neurol., 59 (1934) 375--429. 14 Honig, M.G. and Hume, R.I., Dil and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing, Trends Neurosci., 12 (1989) 333-341. 15 Johnson, J.K., Jinks, R.N, and Chamberlain, S.C., Transneuronal diffusion of DiI in the visual system of the horseshoe crab, Limulus, Soc. Neurosci. Abstr., 16 (1990) 53. 16 Klein, D.C. and Weller, J.L., Indole metabolism in the pineal gland: a circadian rhythm in N-acetyltransferase, Science, 169 (1970) 1093-1095. 17 Lewy, A.J., Sack, R.L., Miller, L.S. and Hoban, T.M., Antidepressant and circadian phase-shifting effects of light, Science, 235 (1987) 352-353. 18 McConneU, S.K., Ghosh, A. and Shatz, C.J., Subplate neurons pioneer the first axon pathway from the cerebral cortex, Science, 245 (1989) 978-982. 19 Moore, R.Y., Organization and function of a central nervous system circadian oscillator: the human suprachiasmatic nucleus, Fed. Proc., 42 (1983) 2783-2789. 20 Moore, R.Y., Retinohypothalamic projection in mammals: a comparative study, Brain Research, 49 (1973) 403-409. 21 Moore, R.Y., The anatomy of central neural mechanisms regulating endocrine rhythms. In D. Krieglar (Ed.), Endocrine Rhythms, Raven, New York, 1979, pp. 63-87. 22 Moore, R.Y. and Eichler, V.B., Loss of circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat, Brain Research, 42 (1972) 201-206. 23 Moore, R.Y. and Klein, D.C., Visual pathways and the central neural control of a circadian rhythm in pineal serotonin N-acetyltransferase activity, Brain Research, 71 (1974) 17-33. 24 Moore, R.Y. and Lenn, N.J., A retinohypothalamic projection in the rat, J. Comp. Neurol., 146 (1972) 1-14. 25 Moore-Ede, M.C., Czeisler, C.A. and Richardson, G.S., Circadian timekeeping in health and disease, N. Engl. J. Med., 309 (1983) 469-476 + 530-536. 26 Pickard, G.E., The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retino-hypothalamic projection, J. Comp. Neurol., 211 (1982) 65-83. 27 Rosenthal, N.E., Sack, D.A., Carpenter, C.J., Parry, B.L., Mendeison, W.B. and Wehr, T.A., Antidepressant effects of
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light in seasonal affective disorder, Am. J. Psychiatrv, 142 (1985) 163-170. Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 69 (1979) 449-526. Sadun, A.A., Schaechter, J.D. and Smith, L.E.H., A retinohypothalamic pathway in man: light mediation of circadian rhythms, Brain Research, 302 (1984) 371-377. Schaechter, J.D. and Sadun, A.A., A second hypothalamic nucleus receiving retinal input in man: the paraventricular nucleus, Brain Research, 340 (1985) 243-350. Stephan, EK. and Zucker, 1., Circadian rhythms in drinking and locomotor activity are eliminated by hypothalamic lesions, Proc. Natl. Acad Sci. U.S.A.. 69 (1972) 1583-1586. Stephan, EK. and Zucker, 1., Rat drinking rhythms: central visual pathways and endocrine factors mediating responsiveness to environmental illumination, Physiol. Behav., 8 (1972) 315326. Stopa, E.G., King, J.C., Lydic, R. and Schoenc, W.C., Human brain contains vasopressin and vasoactive intestinal polypeptide neuronal subpopulations in the suprachiasmatic region, Brain Research, 297 (1984) 159-163. Swanson, L., Cowan, W.M. and Jones, E.G., An autoradiographic study of the efferent connections of the ventral lateral geniculate nucleus in the albino rat and cat. J. Comp. Neurol.. 156 (1974) 143-164. Thanos, S. and Bonhoeffer, F., Axonal arborization in the developing chick retinotectal system, J. Comp. Neurol., 261 (1987) 155-164. Vanselow, J., Thanos, S., Godement, P., Henke-Fahle, S. and Bonhoeffer, F., Spatial arrangement of radial glia and ingrowing retinal axons in the chick optic tectum during development, Dev. Brain Res., 45 (1989) 15-27. VidaI-Sanz, M., Villegas-Perez, M.P., Bray, G.M. and Aguayo, A.J., Persistent retrograde labeling of adult rat retinal ganglion cells with the carbocyanine dye, Dil, Exp. Neurol., 102 (1988) 92-101. von Bartheld, C.S., Cunningham, D.E. and Rubel, E.W., Neuronal tracing with DiI: decalcification, cryosectioning and photoconversion for light and electron microscopic analysis, J. Histochem. Cytochem., 38 (1990) 725-733. Wizenmann, A. and Thanos, S., The developing chick isthmooptic nucleus forms a transient efferent projection to the optic tectum, Neurosci. Lett., 113 (1990) 241-426.
BRES 24813
297
Short Communications
Labeling of human retinohypothalamic tract with the carbocyanine dye, DiI Deborah I. Friedman 1'2, J. Kelly Johnson 4'*, Regina L. Chorsky 3 and Edward G. Stopa 3'5 Departments of 1Neurology, 2Ophthalmology and 3pathology [Division of Neuropathology], SUNY Health Science Center, Syracuse, NY 13210 (U.S.A.), 41nstitutefor Sensory Research, Syracuse University, Syracuse, NY 13210 (U.S.A.) and 5Brain Tissue Resource Center, McLean Hospital, Harvard Medical School, Belmont, MA 02178 (U.S.A.)
(Accepted 28 May 1991) Key words: Retinohypothalamic tract; Suprachiasmatic nucleus; Hypothalamus; Carbocyanine dye; DiI; Circadian rhythm
The carboeyanine dye, DiI, was used to demonstrate human retinohypothalamic tract (RHT) projections in 6 normal human postmortem brains. In 5 of 6 brains, labeling was seen extending from the site of implantation in the distal optic nerve to both the ipsilateral and contralateral suprachiasmatic nuclei. This study confirms the presence of RHT projections in humans, and demonstrates the usefulness of DiI for neuronal tracing in human postmortem tissue. The suprachiasmatic nucleus (SCN) in the anterior hypothalamus has been found to be important for synchronizing circadian rhythms to the environmental light-dark cycle 19'21'28'31. Early studies in rodents demonstrated that afferent visual pathways were necessary for relaying information regarding environmental illumination to the SCN. Bilateral transection of the optic nerve was found to produce desynchronization of circadian rhythmicity, whereas transection distal to the optic chiasm did not alter circadian regulation 16'22'23'31. These observations suggested the presence of a direct retinofugal pathway to the SCN that terminated near the optic chiasm, the retinohypothalamic tract (RHT). Subsequent anatomical studies in numerous species have confirmed that the SCN receives direct retinal projections by way of the R H T 2A3'2°'24'26. In addition, a second indirect retinal projection has been identified from the intergeniculate leaflet, the geniculohypothalamic tract 34. In humans, definitive demonstration of the R H T has n o t been feasible using standard neuroanatomical tracing techniques that generally require viable tissue. However, R H T projections to the human SCN and paraventricular nucleus have been described using the paraphenylene diamine (PPD) technique in patients with long-standing monocular optic nerve damage 29'3°. The utility of the PPD technique is derived from its ability to stain degenerated axon remnants that have persisted in postmortem tissue for long periods of time after the ini-
tial lesion has occurred. Therefore, PPD is a useful anatomical marker of afferent visual pathways when a discrete optic nerve lesion is present, but it cannot be used to examine neuronal pathways in normal brains. Carbocyanine dyes are lipophilic fluorescent substances that provide anterograde and retrograde staining of neurons in vivo and in vitro 35. When applied to brain tissue or peripheral nerve endings in vivo, the dyes are taken up by axons or axon terminals and actively transported to cell bodies 3'14. In fixed tissue, they become inserted into the lipid fraction of the plasma membrane and diffuse freely down the concentration gradient. Labeling has been demonstrated in aldehyde-fixed neuronal tissue in several species 9'1°'12A8'38. DiI (1,1"-dioctadecyl3,3,3",3"-tetramethylindocarbocyanine perchlorate; Molecular Probes, Eugene, OR) has proven to be useful for studying the visual system in various submammalian and mammalian species 4'9'10'15'35-37'39. In the present study we used DiI to confirm the presence of retinal projections to the SCN in normal human brains. Six adult human brains were obtained within 24 h of death. All brains were confirmed to be free of disease by both clinical history and postmortem neuropathological examination. A block of tissue containing the optic nerves, optic chiasm, and hypothalamus was dissected from the fresh brain, and immersed in 4% paraformaldehyde in 0.1 M Sorensen's phosphate buffer (pH 7.2). Some of the brains were briefly immersed in 10% for-
* Current address: Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-2715, U.S.A. Correspondence: E.G. Stopa, Department of Pathology, SUNY Health Science Center, 750 East Adams Street, Syracuse, NY 13210, U.S.A.
298
Fig. 1. Dil crystals implanted into the left optic nerve of a human brain, fixed in 4% paraformaldehyde.
malin after brain removal, and the specimens were placed in 4% p a r a f o r m a l d e h y d e following dissection. Within 24-48 h of death, DiI crystals were e m b e d d e d into the distal end of one optic nerve using an operating microscope, after excess solution had been blotted (Fig. 1). The tissue was then carefully placed back into 4% phosphate-buffered p a r a f o r m a l d e h y d e , and maintained at 37 °C in darkness. A f t e r approx. 3 - 4 months, the hypothalamus was sectioned coronally at 60-100/~m using a vibratome. The sections were collected on gelatin-coated slides, m o u n t e d in an aqueous mounting m e d i u m (Gelmount, B i o m e d a Corp., Foster City, C A ) , and photographed with a fluorescence microscope (Nikon Optiphot, O l y m p u s Vanox) using a standard r h o d a m i n e
filter set. To ensure the long-term stability of the DiI label, the slides were stored at 4 °C in darkness whenever they were not being viewed. In 2 additional h u m a n hypothalami, 50-/~m coronal sections through the SCN were processed for vasoactive intestinal polypeptide (VIP) immunocytochemistry and osmium stained in accordance with p r o c e d u r e s previously described 2. In 5 of the 6 brains, DiI labeling was found to diffuse from the site of implantation in the optic nerve through the optic chiasm, to both the ipsilateral and contralateral SCN (Figs. 2-4). These labeled axonal projections e n t e r e d the ventral surface of the SCN in a manner similar to that described in rodents 2°'24"26. L a b e l e d fibers were also seen decussating within the optic chiasm (Fig.
Fig. 2. Composite montage of DiI fluorescence within the optic chiasm and rostral preoptic hypothalamus, 4 months after the implantation of DiI into the left optic nerve (rhodamine filter). Note the projections to the ipsilateral (arrow) and contralateral suprachiasmatic nuclei (SCN). Some optic nerve fibers can be seen decussating within the chiasm. Fig. 3. Fluorescent fibers can be seen crossing beneath the third ventricle to the contralateral SCN. Fig. 4. High magnification view demonstrating numerous fluorescent fibers within the ipsilateral SCN (top half of picture), arising from the optic chiasm (lower half of picture). The third ventricle can be seen on the left edge of the photograph. Fig. 5. High magnification views of fluorescent fibers within the optic chiasm. Individual fibers can be readily traced as they course across the microscopic field.
v-
ii,,
~i ¸
301 5). Fibers leaving the chiasm en route to the ipsilateral SCN were generally well defined, and their orientation correlated with vertically oriented myelinated fibers observed in this region on osmium-stained preparations (Fig. 6A,B). Correlations between osmium/VIP stained sections and DiI preparations made at comparable anatomic levels confirmed that the DiI labeled fibers were entering the SCN. The SCN is the only site in the anterior hypothalamus that contains a VIP immunopositive neuronal subpopulation 33. Several factors were found to contribute to the overall effectiveness and utility of carbocyanine tracers in postmortem human brain. The rate of DiI labeling was enhanced when the specimens were stored at 37 °C rather than at r o o m temperature. Even under optimum conditions (37 °C with 4% paraformaldehyde fixative), we observed a diffusion rate of only 10 m m after 3 months. Whether or not premortem events also play a role in modulating diffusion rates remains to be determined. The DiI label was more robust in some brains than others, and in one brain it failed to diffuse. Although it is impossible to determine the reason for failure of DiI labeling in this brain with certainty, we speculate that the brief period of initial 10% formalin fixation may be an important contributing factor. Formalin-fixed tissue was generally found to be less suitable than tissue fixed in 4% paraformaldehyde, possibly due to the impurities present in many commercially obtained formalin preparations. After tissues were sectioned, mounted, and viewed, DiI began to fade within approx. 72 h of mounting unless the slides were stored at 4 °C in darkness. Disappearance of the DiI label has been described in association both for the avidin-biotin and peroxidase-antiperoxidase methods, as well as during the incubation of tissue in 0.04% hydroperoxide or 1% sodium borohydride 8.
O u r findings are in agreement with the previous PPD analysis by Sadun and coworkers 29, and confirm the presence of both ipsilateral and contralateral projections from the retina to the ventral human SCN. The improved visualization made possible by the DiI technique provides additional evidence that R H T pathways may be important in mediating various clinically observed phenomena 25. For example, abnormalities of the neuroendocrine regulatory system have been attributed to the absence of diurnal light-dark input in blind persons. Blind women experience earlier menarche than sighted women, and have differences in water, insulin, and carbohydrate balances compared to sighted individuals 11. Manipulation of the light-dark cycle has been shown to shift human circadian rhythms, and has been useful in the treatment of affective disorders 17'27. Additionally, it has been shown that light is a potent entraining stimulus for human circadian rhythms 7, and that exposure to bright light can reset the human circadian pacemaker independently of the sleep-wake cycles 5. Light exposure has also been shown to improve adaptation from daytime to nighttime shift work 6. In conclusion, this study provides further evidence for the existence of the R H T in normal human brains, and demonstrates the usefulness of DiI for neuronal tracing in human postmortem tissue. Future applications of the DiI technique may provide new insights into the anatomy and development of the human nervous system, which were not previously feasible using classical neuroanatomical tracing techniques.
1 Anthony, E.L.P. and King, J.C., Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system, Am. J. Anat., 175 (1986) 179-195. 2 Cassone, V.M., Speh, J.C., Card, J.P. and Moore, R.Y., Comparative anatomy of the mammalian hypothalamic suprachiasmatic nucleus, J. Biol. Rhythms, 3 (1988) 71-93. 3 Catsicas, S., Thanos, S. and Clarke, P.G.H., Initial imprecision in the topography of the chick embryo's isthmo-optic projection, Soc. Neurosci. Abstr., 12 (1986) 984. 4 Collelo, R.J. and Guillery, R.W., The early development of retinal ganglion cells with uncrossed axons in the mouse: retinal position and axonal course, Development, 108 (1990) 515523. 5 Czeisler, C.A., Allan, J.S., Strogatz, S.H., Ronda, J.M., Sanchez, R., Rios, C.D., Freitag, W.O., Richardson, G.S. and Kronauer, R.E., Bright light resets the human circadian pacemaker independently of the timing of the sleep-wake cycle, Science, 233 (1986) 667-671. 6 Czeisler, C.A., Johnson, M.P., Duffy, J.E, Brown, E.N.,
Ronda, J.M. and Kronauer, R.E., Exposure to bright light and darkness to treat physiologic maladaptation to night work, N. Engl. J. Med., 322 (1990) 1253-1259. Czeisler, C.A., Kronauer, R.E., Allan, J.A., Duffy, J.F., Jewett, M.E., Brown, E.N. and Ronda, J.M., Bright light induction of strong (Type O) resetting of the human circadian pacemaker, Science, 244 (1989) 1328-1333. Elberger, A.J. and Honig, M.G., Double-labeling of tissue containing the carbocyanine dye DiI for immunocytochemistry, J. Histochem. Cytochem., 38 (1990) 735-739. Godement, P., Salaun, J. and Mason, C.A., Retinal axon pathfinding in the optic chiasm: divergence of crossed and uncrossed fibers, Neuron, 5 (1990) 173-186. Godement, P., Vanselow, J., Thanos, S. and Bonhoeffer, E, A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue, Development, 101 (1987) 697-713. Hasan, W., Chandra, P. and Prasad, J.N., Continuous illumination and oogenesis in albino rabbits, Ind. J. Physiol. Pharmacol., 25 (1981) 279-284.
The authors wish to acknowledge Drs. Stephen Chamberlain, Andrew Satlin, George Collins and Ellion Albers for many helpful suggestions, and Dr. Edward Bird for providing some of the tissues used in this study. Supported by A ~ 9 5 and AG09301 (E.G.S.), Brain Bank Grant MI-I/NS31862, SUNY Health Science Center Pathology Medical Service Group (E.G.S.) and the Hendricks Fund for Medical Research (D.I.E, E.G.S.).
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