Contains reports on two research projects.National Institutes of Health (Grant 5 PO1 GM14940-07

Gruberg, E. E.

Stirling, Victoria

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X. NEUROPHYSIOLOGYAcademic and Research StaffProf. J. Y. Lettvin Dr. E. R. GrubergProf. S. A. Raymond M. H. BrillGraduate StudentsLynne Galler I. D. Hentall D. W. SchoendorferR. E. Greenblatt M. Lurie Susan B. UdinW. M. SaidelA. AUTORADIOGRAPHIC STUDY OF CHANGES IN THE FROG TECTUMAFTER CUTTING THE OPTIC NERVENational Institutes of Health (Grant 5 PO1 GM14940-07)E. R. Gruberg, R. Victoria StirlingIn adult amphibia, cut optic nerves will grow back to reform precise retino-topic1-3maps onto the tectum. Most theories of mechanisms for the remapping have focusedon the optic fibers playing the active role in reorganizing. The brain has generally beenassumed to remain a passive tissue onto which the optic fibers go about the reconnectingprocess. By default, we had shared this view and we were about to commence on a setof frog tectum manipulations and reversals to test some hypotheses about the manner ofregrowth of optic fibers. But we were aware that our forthcoming experimentswere predicated on the assumption that the brain was well-behaved, i. e., it would notreorganize itself in the course of the experiments. There was one notable report byGaze and Watson 4 describing changes in the tectum of the newt when the optic nervewas cut. At a variety of intervals post-operatively, they had injected animals with3 H-thymidine, and the brains were subsequently processed using autoradiography. Theyfound labeled cells in the tectal neuropil and in the ependymal cell layer. Salamanders,even compared with frogs (let alone higher vertebrates), are, of course, exemplary fortheir regenerative ability. But as a preliminary control, we thought we would repeatand, if necessary, extend the experiments of Gaze and Watson in the frog.Four frogs (Rana pipiens), from 2 in. to 2 1/2 in. long and weighing 20-30 grams,were anesthetized with Finquel (Ayerst). By cutting the appropriate extraocular mus-cles of the left eye, the optic nerve was exposed and cut. The animals were sub-sequently kept in a constant-temperature room (20-21 ° C) and injected subcutaneouslyin the foreleg with 20 C 3H-thymidine (New England Nuclear Corporation) after 8, 11,15, or 20 days. The day after injection the brains were removed after decapitation andfixed overnight in 4% buffered neutral formalin. The brains were then dehydratedthrough a set of graded alcohols and cedarwood oil, embedded in paraffin, serially sec-tioned at 7 [im and mounted on albumenized slides. The sections were deparaffinized inQPR No. 111E/CBI7AEFig. X-1. Camera lucida transverse sections of the midbrain of a frogshowing the distribution of labeled cells (oblong marks). Inset:locations of the sections.(a) After injecting 20 [IC 3H-thymidine 8 days after left opticnerve section. Brain excised on the following day. Eachmark corresponds to one labeled cell.(b) After injecting 20 FC 3H-thymidine 14 days after left opticnerve section near parasphenoid bond. Brain excised on thefollowing day. Each mark corresponds to 3 labeled cells.QPR No. 111ji c142(X. NEUROPHYSIOLOGY)xylene, then hydrated. In total darkness the slides were dipped in Kodak NTB-2nuclear-track emulsion diluted 1:1 with water warmed to 400 C, dried for 1 hour, andplaced in light-tight slide boxes in a refrigerator (40 C) for 1 week. The slides weredeveloped in total darkness for 2 min in full strength D-19 (Kodak), rinsed brieflyin acid stop, and fixed (Kodak fixer) for 6 min. The slides were washed in running waterfor 20 min, stained for 10 min in 0. 1% cresyl violet, dehydrated, and mounted withcoverslips.We found that after 8 and 11 days, labeled cells were distributed homogeneously inoptic fiber layers of the right (contralateral) tectum. The left tectal lobe had only afew labeled cells, but they were distributed in the retinal fiber layers. Eight and 11 daysafter cutting the optic nerve significant degeneration is seen in the tectal retinal fiberlayers (Fig. X-la). It suggests that the labeled cells could be newly formed macro-phages concerned with clearing away the debris of optic fiber degeneration.After 15 days, labeled cells are not seen in retinal fiber layers of the contralateraltectum, but the ependymal cell layer is replete with them (Fig. X-1b). The labeledcells are not distributed uniformly in the ependymal layer but are found for the most partin the postero-lateral area. No labeled cells are seen in the ipsilateral tectum. Indevelopment, the cells of this layer generate all the cells of the tectum, which thenmigrate out to their final locations. No labeled cells were found after 20 days.The distribution and times of formation of the labeled ependymal cells were studiedfurther. In a set of animals, the optic nerve was cut and, using the same procedure,we injected with 20 [iC 3H-thymidine after 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 days. Only at 15 and 16 days did we find labeled ependymal cells. The dis-tribution was identical with that described.In the next set of animals, a slit was made in the roof of the mouth, and a smalllength of the optic nerve was cut just outside its entry into the parasphenoid bone. Ani-mals were injected with 20 C 3H-thymidine after 4, 6, 8, 10, 12, 14, 15, 16, 17, or18 days. The eyes and brain were removed the next day. Since the nerve was cut closerto the brain, ependymal cells were labeled earlier, but only on days 13 and 14(Fig. X-2). The distribution was the same as described above. Except for the ciliarymargin, few labeled cells were seen in the retina at any time, but many labeledcells were found in the optic disc on days 12, 13, and 14 (Fig. X-3a, 3b).We next looked at the long-term fate of the labeled ependymal cells: Would theymigrate out to neural layers, and would that depend upon the reacquisition of vision ofthe eye whose optic nerve had been cut? Each animal was injected with 20 IC3H-thymidine 13, 13 1/2, and 14 days after the left optic nerve was cut. They were thentransferred to a warm, sunny room (average temperature approximately 23 -240 C) andfed 6-10 crickets a week. Animals were tested behaviorally for vision by a sweepingmotion with the hand from the left or right side. After the optic nerve was cut, theQPR No. 111 143Fig. X-2. (a) Superficial tectum showing distribution of labeled cells.3 H-thymidine injected 8 days after optic nerve section.Brain excised on the following day. Scale: 50 pm mag-nific ation.(b) Periventricular area showing distribution of labeledcells. Thymidine injected 14 days after left optic nervesection. Brain excised on the following day. Scale:same as in (a).(c) and (d) Refer to (D) in Fig. X-lb. Same section as in(a) showing ipsilateral (c) and contralateral (d) tectum.Black mark in the middle of the right area of the tec -tum (d) is a pigment granule. Scale: 200 p.m.QPR No. 111 1440(Scale: 50 m.tral is up. Scale: same as in (a).3H-thymidine 13, 13 1/2, and 14 days after optic nervemonths. At the time of brain excision, animal (c) did not(a)ec ion of eye throughblind eye, no optic disc (D) and adjacenretina (R) showing ccumulation of labeled cells in thlayer.Animal (d) had recovercted vision 2 1/2 weeks earlic nerve, andsetion. Eyed cells have migrated on the foll(arrow).ing day.e:Scale: 50 m.m.some labeled cells have migrated out (arrows). Scale:QPR No. 111d d:~-l~a0145(X. NEUROPHYSIOLOGY)animals would jump away from a hand moved toward them from the right side but notfrom the left side. In several animals we waited as long as six months but there wasno return of vision. In these animals, when the brain was removed, no left optic nervewas present. Labeled cells could still be seen in the ependymal cell layer, but nonewere seen in higher layers. In two animals whose optic nerve was cut cleanly, visionreturned within 9 weeks so that the frog would jump away from a hand moved in fromeither direction. An observer who did not know which optic nerve had been cut couldnot distinguish between responses from the left or right side. The brains were removed17 days later (or 3 1/2 months after the optic nerve was cut), and the labeled cellswere found to have migrated out to neural layers. Most labeled cells were still in theependymal layer, but in each section a handful of cells could be seen to have migratedout (Fig. X-3c, 3d).We have thus shown in the adult frog that (i) New ependymal cells are formed forthe most part in the contralateral-posterolateral area of the tectum after the optic nerveis cut. (ii) They are formed only during a brief interval of time - 13-14 days aftercutting the optic nerve via the upper mouth and maintaining the animals at 20 -21' C.(iii) Some of these cells migrate out to neural layers only in animals that havereacquired some vision.It remains to be shown whether these migrating cells are neurons or glia. The cri-teria for distinguishing between them in light microscopy has been described as inade-quate by several authors.5, 6 But several points are clear. The localization of the labeledcells along the tectal ventricle is similar to that found in late development.7 Roughly, theanterior tectum is created and developed first and the caudal part last.8 In the absenceof optic fiber input, it appears that the tectal lobe has regressed to a late embryonicstate. This would suggest that at least some of the migrated cells are neurons. Butthe answer to this will await future results of experiments in progress.References1. H. R. Maturana, J. Y. Lettvin, W. S. McCulloch, and W. H. Pitts, "Evidence thatCut Optic Nerve Fibers in a Frog Regenerate to Their Proper Places in the Tectum,"Science 130, 1709 (1959).2. R. M. Gaze and M. A. Jacobson, "A Study of the Retinotectal Projection duringRegeneration of the Optic Nerve in the Frog," Proc. Roy. Soc. (London) Ser. B157, 420-480 (1963).3. J. Cronly-Dillon, "Pattern of Retinotectal Connections after Retinal Regeneration,"J. Neurophys. 31, 410-418 (1968).4. R. M. Gaze and W. E. Watson, "Cell Division and Migration in the Frog," inG. Wolstenholme and M. O'Connor (Eds.), Growth of the Nervous System (Little,Brown and Co., Boston, 1968), pp. 53-67.5. G. R. DeLong and R. Sidman, "Effects of Eye Removal at Birth on Histogenesis inthe Mouse Superior Colliculus: An Autoradiographic Analysis with Tritiated Thy-midine," J. Comp. Neurol. 118, 305-344 (1962).QPR No. 111 146(X. NEUROPHYSIOLOGY)6. J. Altman, "Autoradiographic and Histological Studies of Post-natal Neurogenesis.IV. Cell Proliferation and Migration in the Anterior Forebrain with Special Refer-ence to Persisting Neurogenesis in the Olfactory Bulb," J. Comp. Neurol. 137, 433-458 (1969).7. K. Straznicky and R. M. Gaze, "The Development of the Tectum in Xenopus laevis:An Autoradiographic Study," J. Embryol. Exp. Morph. 28, 87-115 (1972).8. R. M. Gaze, S-H. Chung, and M. J. Keating, "Development of thejection in Xenopus," Nature New Biology 236, 133-135 (1972).Retinotectal Pro-B. PRESENCE OF MONOAMINES IN THE SALAMANDER TECTUMAND THEIR RELATION TO THE SPINAL LEMNISCUSNational Institutes of Health (Grant 5 PO1 GM14940-07)E. R. GrubergThe salamander tectum is a cylindrical structure monopolizing the dorsal mesen-cephalon. There is a ventricle in the center (the "aqueduct"), several rows of cellsperiventricularly, and an outer neuropil free of cell bodies but composed of a dense net-work of dendrites from the periventricular cells and incoming axons from a variety ofFig. X-4. (a) Section through thoracic spinal cord showing scatteredfluorescence.(b) Section near rostral border of spinal cord where thediscrete lateral fluorescent tract is first encountered.(c) Same tract as (b) in vagal region.(d) Same tract as (b) in rostral medulla. Upper right:nerve V.QPR No. 111Ii147(X. NEUROPHYSIOLOGY)sources. The outer half of the neuropil is replete with optic nerve fibers forming aretino-topic projection. Using histochemical techniques, we have shown previously thatthese fibers contain acetylcholinesterase. The boundary of the enzyme is sharp, and theinner half of the neuropil shows no acetylcholinesterase activity. This inner area con-tains numerous somesthetic fibers and they are organized to form a coarse but recog-nizable map of the contralateral body that is in register with the retino-tectal map. Itwas of interest to see if there are potential neurotransmitters that could be correlatedwith the somesthetic input. We knew that SSADH, the final enzyme in the degradativepathway of GABA, was not localized to this region, so another set of possibilities werethe monoamines. We used the standard histo-fluorescent method to show the distribu-tion of monoamines in the brain of the tiger salamander.Brains were removed rapidly from the salamanders after decapitation and frozenquickly in 2-methylbutane cooled by liquid nitrogen. The brains were transferred to afreeze drier kept at -40 C and dried until the pressure was reduced to 0. 01 Torr(approximately 1-3 days). The brains were then fixed at 80 0 C for 1 hour over paraform-aldehyde that had in turn been dried overnight over concentrated sulfuric acid. Thefixed brains were vacuum-embedded in paraffin in 2 minutes. Transverse sections(10 tm) were cut just prior to viewing in a dark-field fluorescence microscope. Fluo-rescent tissue stood out as bright green-yellow on a dark blue-green background. Differ-ent monoamines fluoresce at different wavelengths, but without a microspectrophotometersubjective judgments about the wavelengths are erratic. Therefore, for purposesof this description, no distinction between monoamines is made. Since the half-life ofthe fluorescence under the ultraviolet illumination used in our microscope was less than1 min, sections were scanned rapidly and photographed immediately on Type 47 Polaroidfilm (3000 speed). Exposure times were 20-40 seconds.In the upper spinal cord, fluorescence is scattered through most of the neuropil.Only at the border of the medulla is a well-defined tract seen on the lateral wall. Thistract can be followed through the whole length of the medulla to the level of the cerebel-lum (Fig. X-4). The tract splays out through the caudo-dorsal tegmentum into the innerneuropil of the tectum. The whole course of the tract is coincident with Herrick'sdescription of the spinal lemniscus 2 (this was also checked by using degenerationstaining). The fluorescence does not extend into the outer neuropil, and in favorablesections the optic fiber area is a dark wedge silhouetted by surrounding fluorescence(Fig. X-5). With notable exceptions, very few cell bodies fluoresce. Some scatteredfluorescing cells are seen in the spinal cord. The medial cells of the nucleus interpe-duncularis are moderately fluorescent. The most vividly fluorescent nucleus is locatedin the region between the dorsal and ventral hypothalamus. In the anterior preopticnucleus some cells lining the preoptic recess also show vivid fluorescence (Figs. X-6and X-7).QPR No. 111 148Fig. X-5. (a), (b), and (c) Section of tectum showing fluorescent fibersin inner neuropil with no fluorescence in outer neuropil.(d) Fluorescence in dorsal tegmentum extending up to tectum.(e) Cellular fluorescence in nucleus interpeduncularis.Fig. X-6. (a)(c)(d)and (b) Fluorescence in a lamina of the hypothalamus.Fluorescence in a ventral thalamic nucleus.Fluorescence in cells of the anterior part of thepreoptic nucleus.QPR No. 111 149Fig. X-7. Sketches of transverse spinal cord and brain sections with numberedboxes showing approximate locations of the photomicrographs inFigs. X-4 through X-6.QPR No. 111 150_~ lit LL-- I I -h-~h~ -- ---- F~-- ~--Fig. X-8.(a) Rostral spinal cord showing degen-eration ipsilateral to more caudalhemisection. 10-day degeneration.(b) Same as (a) showing contralateralarea.(c) Two branches of spinal lemniscus.Lower center branch referred to by2Herrick as bulbar lemniscus. Sameanimal as (a).(d) Ipsilateral to cord hemisection ofanother animal showing degenerationin dorsal tegmentum and extendinginto tectum. 12-day degeneration.Fig. X-9.Al; Tectum caudal to oculomotor nucleussho wing degeneration restricted toinner neuropil. (a) Ipsilateral to cordhemisection and (b) contralateral. 10-day degeneration. (c) and (d) Sectionsin ipsilateral cerebellar areas androstral medulla showing pattern of 10-day degeneration.QPR No. 111'~' -I~ERZTI-~-I~.L1151(X. NEUROPHYSIOLOGY)8a 8b8c.9 9b9jFig. X-10. Camera lucida drawings of transverse sections showing distributionof 10-day degeneration. Thoracic spinal cord hemisection of leftside. Numbered boxes show locations of the photomicrographs inFigs. X-8 and X-9.To check for the course of the spinal lemniscus, hemisection of the spinal cord wasmade in several salamanders (all approximately 4 in. snout to vent). Then by a modifi-cation of the Fink-Heimer stain for degenerating nerve fibers, the lemniscal pathway wastraced. Under Finquel (Ayerst) anesthetic, one vertebra in the upper thoracic regionwas removed in exposing the spinal cord. Hemisections were made with a scalpel, andthe animals were maintained postoperatively at 20-21"C for 6, 8, 10 or 12 days. Thetissue was then processed for degeneration staining.Degeneration rostral to the lesion is scattered throughout the neuropil. It is heavieripsilaterally than contralaterally. By the caudal medulla the spinal lemniscus emergesas a distinct tract and can be followed the length of the medulla (Fig. X-8). Some fibersterminate in a narrow band in the cerebellum, but many fibers continue and end bilater-ally in the inner half of the tectal neuropil (Fig. X-9). Little degeneration is seen in thetectum rostral to the level of the oculomotor nucleus, although more ventral degenera-tion can be traced up to the ventral thalamus. The degeneration of one branch of thespinal lemniscus through its whole length and into the tectum corresponds directly withthe fluorescent tract seen above. A second more medial branch (not fluorescent) of thelemniscus, which Herrick 2 assumed was of bulbar origin, can be followed to the uppermedulla (Fig. X-10).The fluorescence in the inner neuropil of the tectum extends farther rostral than thelemniscal degeneration. From electrical recordings it is known that this area has somes-thetic input from the head region. Thus contributions to the rostral tectal fluorescencemust be made from cranial sources.QPR No. 111 152(X. NEUROPHYSIOLOGY)References1. E. R. Gruberg and G. A. Greenhouse, "Synthesis of Acetylcholine and Gamma-Aminobutyric Acid in the Tectum of the Tiger Salamander, Ambystoma tigrinum,"Quarterly Progress Report No. 110, Research Laboratory of Electronics, M.I. T.,July 15, 1973, pp. 185-188.2. C. J. Herrick, The Brain of the Tiger Salamander (University of Chicago Press,Chicago, 1948).QPR No. 111 153

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