Intraocular injections of tetrodotoxin reduce transiently expressed acetylcholinesterase activity in developing rat visual cortex

Developmental Brain Research, 46 (1989) 69-84

69

Elsevier BRD50873

Intraocular injections of tetrodotoxin reduce transiently expressed acetylcholinesterase activity in developing rat visual cortex Richard T. Robertson 1, Rajeev K. A m b e 1 and Jen

Yu 2

Departments of 1Anatomyand Neurobiology and 2physicalMedicine and Rehabilitation, Collegeof Medicine, University of California, Irvine, CA 92717(U.S.A.) (Accepted 11 October 1988)

Key words: Acetylcholinesterase;Cerebral cortex; Development; Tetrodotoxin; Visual deprivation; Visual system

Geniculo-recipient layers of primary visual cortex in the rat display a transient pattern of acetylcholinesterase (ACHE) activity during the second postnatal week of life. Previous work has demonstrated that neonatal enucleations markedly reduce the transient AChE activity in visual cortex. The present studies were undertaken to determine the effects of reduced afferent neural activity on expression of the transient pattern of AChE activity. Rat pups received intraocular injections of tetrodotoxin (TTX) on postnatal days (PND) 3, 5, 7, 9 and 11 and were sacrificed on PND 12. Some animals were enucleated on PND 3. Brain sections were processed for AChE histochemistry and analyzed by optical densitometry. These experiments show that uniocular injections result in a markedly decreased level of AChE activity in layer IV of the medial part of cortical area 17 contralateral to the injected eye. The degree of reduction of AChE activity from repeated TTX injections was similar to the degree of reduction following enucleation on PND 3. Binocular injections of TI'X result in a reduction of AChE activity in layer IV throughout cortical area 17, similar to the effects of binocular enucleation on PND 3. Experiments combining injection of horseradish peroxidase along with TTX on PND 11 demonstrate that retinal ganglion cells of TI'X injected eyes are still capable of anterograde axonal transport. These data demonstrate that normal innervation and afferent activity are necessary for the transient expression of AChE activity by geniculocortical neurons. INTRODUCTION R e c e n t work from this l a b o r a t o r y has revealed the presence of an intense and transient pattern of acetylcholinesterase (ACHE) activity in cortical area 17 of the developing l a b o r a t o r y rat 43-49. This p a t t e r n of A C h E first appears in cortical area 17 at about postnatal day 6 ( P N D 6), reaches p e a k intensity about P N D 10-12, and then declines to adult levels by P N D 2143,44,49. Within cortical area 17, the transient A C h E appears as a dense, fiber-like plexus primarily in cortical layer IV and the deepest portion of layer IIl, thus corresponding to the terminal zone of geniculocortical projections 35,37,67. The close correspondence between the distribution of transiently expressed A C h E and the location of geniculocortical terminal fields suggests that the transient A C h E may serve as a m a r k e r for geniculocortical axon termi-

nals. This suggestion receives support from experiments that d e m o n s t r a t e loss of transient A C h E in cortical area 17 following placement of lesions in the dorsal lateral geniculate nucleus ( d L G N ) 45. O t h e r recent work from this l a b o r a t o r y has indicated that this transient p a t t e r n of A C h E is experimentally manipulable. That is, neonatal removal of one eye results in an absence of the transient pattern of A C h E in the medial (monocular) region of cortical area 17 in the hemisphere contralateral to the enucleated orbit 44,49. Further, neonatal bilateral eye removal results in an absence of transiently expressed A C h E throughout area 17 of both hemispheres 44. These data indicate that normal innervation or neural activity is necessary for the normal d e v e l o p m e n t of the transiently expressed ACHE. The results from experiments employing neonatal eye removal are somewhat difficult to interpret, how-

Correspondence: R.T. Robertson, Department of Anatomy and Neurobiology, College of Medicine, University of California, Irvine, CA 92717, U.S.A. 0165-3806/89/$03.50 (~) 1989 Elsevier Science Publishers B .V. (Biomedical Division)

70 ever, because the enucleation results not only in an absence of normal afferent input to the dLGN, but also rapid degeneration of the optic tract with concomitant loss of possible trophic factors provided to the neurons of dLGN by the optic tract. An alternative means of depriving the dLGN of optic input is to administer tetrodotoxin (TTX) to the retina 8'1°'32' 34.39-~1.59. TTX is a toxin that blocks sodium channels (including those in retinal ganglion cells) and thus eliminates action potentials. Thus, repeated intraocular administration of TTX offers a means to eliminate optic tract input to the neurons of dLGN without necessarily causing confounding degeneration. This report describes our experiments studying the effects of repeated administration of TTX on development of transient AChE activity in visual cortex. Portions of these data have been presented previously in abstract form 1. MATERIALS AND METHODS

Animals. Experiments were performed on approximately 58 Sprague-Dawley rat pups from 8 different litters. Pregnant dams were purchased from licensed breeders and pups were born in the laboratory. Litters were adjusted to 8 pups for each dam on the day of birth (PND 0). Surgical procedures. Rat pups received repeated intraocular injections of TTX (0.5 ktl; 10 .4 M in sodium-phosphate buffered saline) either unilaterally or bilaterally on PND 3, 5, 7, 9 and 11. Animals were lightly anesthetized prior to injections by hypothermia on PND 3 and 5 and by ether on PND 7, 9 and 11. The primordial palpebral fissure was identified under a surgical microscope and opened surgically. The surface of the eye was rinsed with a solution containing 2% procaine, 1% Furacin, and 20% ethanol in water. With the aid of the surgical microscope, the tip of a 30-33 gauge needle attached to a microsyringe was inserted through the sclera. The needle was inserted just posterior to the limbus and penetrated into the vitreous body to a depth of approximately 2 mm. The TTX was injected and the needle held in place for about 1 min. The surface of the eye was rinsed again after the injection. Effectiveness of the TTX was evaluated by injecting a comparable volume of 10 -4 M TTX in the vitreous body of anesthetized juvenile (50-60 g) rats. Pupillary response to variation in in-

tensity of light reaching the retina was used to determine the effect of TTX on optic nerve activity. This assay was used because the pupillary reflex is difficult, at best, to test in infant rats. Some littermates of TTX-injected animals were enucleated either unilaterally (5 animals) or bilaterally (4 animals) on PND 3. These animals were anesthetized by hypothermia, the eye removed, and the enucleated orbit packed with Gelfoam. Topical antibiotics and local analgesics were administered. Seven animals that received the schedule of TTX injections also received intraocular injections of 0.5 /A 30% horseradish peroxidase (HRP) on PND 11. Normal animals and animals receiving sham surgeries served as controls. All animals were sacrificed on PND 12. Animals were deeply anesthetized by chloral hydrate or sodium pentobarbital and perfused through the heart with an aqueous solution of 10% formalin in 0.1 M sodium phosphate buffer at pH 7.3. Animals with H R P injections were perfused with saline, followed first by a fixative solution containing 1% paraformaldehyde and 2% glutaraldehyde in sodium phosphate buffer and second by a solution of 10% sucrose in buffer. Each brain was inspected carefully for the presence of optic nerves as they were removed from the skulls. Brains from all animals were stored in 30% sucrose in buffer overnight at 4 °C. Histochemistry. Frozen sections of 40-64 ktm were cut either in the transverse plane or parallel to the pial surface after the cortex had been removed from the rest of the brain and flattened between glass slides. Transverse sections were collected in batches of 3 and a 1-in-3 series was processed for AChE histochemistry as described below; adjacent sections were processed for a Nissl stain. In cases with H R P injections, alternate sections were processed for H R P histochemistry. Every section from flattened cortices was processed for AChE histochemistry. It is important to point out that tissue was processed as 'sets' in which direct comparisons could be made between tissue from littermates that were TTX-treated or shamoperated control animals. Further, littermates were used for comparisons between enucleated and TTXtreated animals. Tissues from each 'set' of animals were processed through the same fixative and histochemical steps. Free floating sections were processed for AChE

71 histochemistry using a modified version of the method of Koelle and Friedenwald 5'2°. The substrate was 1.0 x 10-4 M acetylthiocholine iodide and non-specific cholinesterase was inhibited by 1.14 x 10 -4 M iso-OMPA5. Sections were incubated for 20-36 h at room temperature. The histochemical reaction product was developed by placing the sections in an aqueous solution of 1% ammonium sulfide for 30 s. The histochemical reaction product in some cases was intensified with 1% silver nitrate. Material presented in the photomicrographs and analyzed by densitometry was not silver-intensified. Sections were dehydrated, coverslipped and examined under the light microscope. Relative density of AChE histochemical reaction product was taken as an index of the relative amounts or activities of AChE 5'6,57,5s. Selected sections were processed for HRP histochemistry using the technique of Mesulam 31. Sections were rinsed and incubated for 20 min in an aqueous solution of 0.05% tetramethyl benzidine and 0.01% hydrogen peroxide. Data analysis. Quantitative assessments of effects of TTX administration and enucleation on AChE activity were made using densitometric analyses. Sections placed on a Zeiss Axiomat microscope were viewed with the aid of a video camera and monitor. The video-recorded image was digitized and analyzed with a deAnza densitometric system. The computer system determined a relative grey value for a rectangular area 500 pixels by 70 pixels (240 × 34 pro). The converted grey value varied from '0' (white) to '250' (black) and indicated the density of AChE histochemical reaction product. Intensity of illumination, contrast of the image and sensitivity of the densitometer were adjusted for each brain so that measurements of AChE reaction product density from the internal capsule would produce readings of 50-60. Densitometric measurements were made starting external to the pial surface and moving orthogonal to the pia through the cerebral cortex to subcortical white matter. Density measurements from two adjacent readings were averaged; this yielded about 14 measurements for each pass through cortex. Two to 6 passes through the cortex were taken for cortical area 17 in each hemisphere and averaged for the data presented in graph form. Most measurements were taken from the middle third 6f visual cortex (in the anterior-posterior di-

mension), although some additional measurements were taken from rostral and caudal levels. Particular attention was paid to comparing the laminar patterns of AChE histochemical reaction product in the two hemispheres and between medial and lateral divisions of area 17 within each hemisphere. Our previous studies of changes in cortical AChE patterns following enucleations 44,49or subcortical ablations 45 indicated that manipulations involving the retino-geniculate system altered AChE staining primarily in cortical layer IV. Thus, differences in the laminar patterns of the two hemispheres would be expected to be greatest in the measurements corresponding to cortical layer IV. The Kolmogorov-Smirnov two-sample test was selected to examine differences in the laminar patterns of AChE of the two hemispheres because it is sensitive to any differences in the cumulative distributions of the two laminar

Fig. 1. Photomicrographsillustrating the pattern of AChE histochemical reaction product in occipital cortex of normal PND 12 rats. Arrowheads indicate borders of cortical area 17. A: AChE-stained transverse section; medial is to the left. Note the dense band of AChE activity in layer IV of cortical area 17. B: AChE-reacted section cut parallel to the pial surface; anterior is to the left, lateral to the top of this photograph. Note the AChE staining throughout area 17. Bar = 500pm for A and B.

72 profiles 56. Any point at which the two measurements are markedly different (i.e. corresponding to cortical layer IV) would be indicated by a difference in the two cumulative distributions and the significance of this difference can be calculated 56.

product appears in visual cortical area 17 of the developing rat as a dense fiber-like plexus primarily in layer IV and the deeper portion of layer Ill. The areal and laminar patterns of AChE can be seen in transverse sections (Fig. 1A) and the areal pattern clearly seen in sections cut parallel to the pial surface (Fig. 1B). AChE staining patterns in the two hemispheres of normal animals appear symmetric. Previous work from this laboratory has demonstrated that this pattern of AChE in cortical area 17 is transient; it first appears at about PND 6, reaches peak

RESULTS

Distribution of A C h E in visual cortex of normal rat pups As shown in Fig. 1, AChE histochemical reaction

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2. Graphic presentation of results of densitometric measures of the laminar distributions of AChE histochemical reaction product through cortical area 17. Cortical depth is represented on the ordinates from outside the piat surface (16) to subcortical white matter (1). Density of AChE reaction product is represented on the abscissa. A: left hemisphere, lateral area 17. B: left hemisphere, medial area 17. C: difference between AChE densities in lateral and medial zones of area 17. D: right hemisphere, lateral area 17. E: right hemisphere, medial area 17. F: difference between AChE densities in lateral and medial zones of area 17. G: difference in AChE densities between hemispheres in lateral area 17. H: difference in AChE densities between hemispheres in medial area 17. Note the peaks of AChE reaction product density in regions corresponding to cortical layer I (just under the pial surface, position 15) and layer I I I - I V (positions 10-12). Note also that AChE peaks in layer III-IV appear greater in lateral than in medial portions of area 17.

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73 intensity at PND 10-12, and declines to adult levels by PND 2143-45'49. The material presented in this report is taken from rat pups sacrificed at PND 12, when the transient pattern of AChE is most intense. The data presented in the photomicrographs of Fig. 1 are corroborated by graphical presentation of densitometric data in Fig. 2. For example, the peaks of AChE activity that correspond to layer I and to layer I I I - I V are clearly seen in the graphs of Fig. 2A,B,D and E. Graphs G and H, presenting differences between AChE staining profiles of the left and right hemispheres, indicate the similarity in staining pattern of the two hemispheres of this normal animal. A null hypothesis would assume zero differences between the density profiles of the two hemispheres. Use of the Kolmogorov-Smirnov test allows us to accept the null hypothesis for both medial and lateral zones of cortical area 17, i.e. the hemispheres show no differences in cumulative AChE density profiles. The two hemispheres show similarity in laminar profiles of AChE staining throughout their rostral-caudal extents. The photomicrographs in Fig. 1 indicate that with-

in layer IV of cortical area 17, AChE staining appears relatively even. However, close observation suggests that lateral portions of area 17 may be slightly more heavily stained than medial portions and such medial-lateral differences are also detected by densitometric analyses. That is, although these graphs demonstrate that the laminar distribution of AChE histochemical reaction product is similar in medial and lateral portions of cortical area 17, the lateral portion displays slightly denser staining in layer I I I - I V . Note that differences between lateral and medial portions of cortical area 17 are greatest for points corresponding to layer I I I - I V (Fig. 2C,F).

Effects of uniocular administration of T T X on the distribution of A C h E in visual cortex Photomicrographs in Fig. 3 present results from a case in which TTX was administered to the right eye of a rat pup on postnatal days 3, 5, 7, 9, and 11, and the animal was sacrificed on PND 12. The ACHEreacted sections in the photomicrographs of Fig. 3A and B illustrate that the density of AChE histochemical reaction product is decreased in the medial por-

Fig. 3. Photomicrographs illustrating effects of unilateral T r x injections on AChE staining in occipital cortex. A,B: transverse AChE-stained sections through the occipital poles illustrating the decrease in AChE staining in medial division of area 17 of the left hemisphere (A), contralateral to the injected (right) eye. C,D: Nissl-stained sections adjacent to sections shown in A and B. Arrowheads indicate borders of area 17. Bar in D = 500/~mfor A-D.

74 tion of cortical area 17 of the left hemisphere, contralateral to the q-TX injections. Note also in Fig. 3 that lateral portions of area 17 of the left hemisphere appear unchanged, i.e. the distribution of A C h E reaction product appears similar to the pattern in the other hemisphere and similar to s h a m - o p e r a t e d littermate control animals. The decrease in density of A C h E staining in medial portions of area 17 contralateral to T T X injections was apparent throughout the rostral-caudal extent of occipital cortex. No change in A C h E staining was detected in the right hemisphere, ipsilateral to the T T X injections. Inspection of the A C h E - r e a c t e d sections in Fig. 3 A and

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Fig. 4. Densitometric data showing effects of unilateral administration of TTX on AChE reaction product density profiles in cortical area 17. A: lateral area 17, contralateral to TTX. B: medial area 17, contralateral to TTX. C: difference between AChE profiles in lateral and medial area 17, contralateral to TTX. D: lateral area 17, ipsilateral to qTX. E: medial area 17, ipsilateral to TTX. F: difference between lateral and medial area 17, ipsilateral to TTX. G: differences between the two hemispheres, lateral portion of area 17. H: differences between the two hemispheres, medial portion of area 17. Symbols as in Fig. 2.

75 A C h E staining is detected as a difference in staining profiles between the two hemispheres (Fig. 4H) and as a greater difference between medial and lateral portions of area 17 in the hemisphere contralateral to T I ' X injections than in the hemisphere ipsilateral to T T X injections (Fig. 4C and F). The K o l m o g o r o v Smirnov test demonstrated that the A C h E density profiles of the two hemispheres differed significantly

for medial parts of area 17 (D = 0.386; P < 0.05) but not for lateral parts of area 17 (D = 0.119; P > 0.2). Results from another case are illustrated in Fig. 5. This series of photomicrographs reveals a reduction in staining of A C h E in medial portions of cortical area 17 contralateral to the T I ' X injections. Further, these photomicrographs demonstrate that the reduction in A C h E staining in medial portions of area 17 is

m Fig. 5. Photomicrographs of a series of AChE-stained, transverse sections through occipital cortex from an animal that received unilateral TI'X injections in the fight eye. Arrowheads indicate the borders of cortical area 17. Note the decrease in density of AChE stain-

ing in medial portions of area 17 of the left hemisphere from anterior (A) through posterior (C) levels. Bar in C = 1 mm.

76 detected from rostral (Fig. 5A) through the caudal (Fig. 5C) extent of area 17. Reduction in A C h E staining in the medial portion of area 17 is also detected in sections cut parallel to the pial surface. In these cases, every section was processed for A C h E histochemistry and Fig. 6 presents photomicrographs of sections showing the maximal areal extent of the ACHE. Note that the relatively even distribution of A C h E reaction product throughout area 17 of the left hemisphere, ipsilateral to the injected eye, is similar to the pattern seen from sham-operated littermate control animals (Fig. 1B). Note further the relative decrease in staining density in the medial portion of area 17 of the right hemisphere, contralateral to the injected eye.

Comparison of effects of monocular administration of TTX and monocular enucleation on the distribution of AChE in visual cortex The photomicrographs presented in Fig. 7 illus-

trate the pattern of A C h E histochemical reaction product in occipital cortices of two littermate rat pups that were subjected to the standard series of unilateral TTX injections (Fig. 7A) or were unilaterally enucleated on PND 3 (Fig. 7B). These animals were sacrificed at the same time and the tissue processed for A C h E histochemistry together. These photomicrographs present the hemispheres contralateral to the experimental manipulation. Note in both photomicrographs the decrease in density of the A C h E reaction product in the medial sector of area 17. Again, this decrease is apparent in cortical layer I l l - I V . The photomicrographs in Fig. 7 suggest that the degree of reduction of A C h E staining in medial portions of area 17 are similar, whether induced by TTX injections or by enucleation. This suggestion from visual inspection is corroborated by densitometric analyses, as summarized in Fig. 8. Fig. 8C and F demonstrate similar differences in A C h E density profiles between medial and lateral portions

Fig. 6. Photomicrographs of AChE-stained tangential sections of occipital cortex from an animal that received unilateral TTX injections in the left eye. Arrowheads indicate the borders of cortical area 17. Note the relatively normal AChE staining of area 17 of the left hemisphere (A) ipsilateral to the injections and the decrease in AChE staining of medial portions of area 17 in the right hemisphere (B) contralateral to the injections. Bar = 500/~m,

77 of area 17 for both cases. Kolmogorov-Smirnov tests on densitometric data from medial regions of left and right hemispheres revealed significant differences between the two hemispheres for both the TTX-injected case (D = 0.337; P < 0.01) and the enucleated case (D = 0.389; P < 0.01). In agreement with other cases reported above, analysis of series of transverse sections reveal that in both cases the decrease in AChE in medial portions of area 17 is found throughout the rostral-caudal extent of area 17. Further, analysis of AChE-stained sections from the hemisphere ipsilateral to the manipulated eyes revealed no difference from the pattern of AChE in sham-operated or normal control animals.

Comparison of effects of binocular administration of TTX and binocular enucleation on the distribution of AChE in visual cortex Fig. 9 presents photomicrographs of ACHEstained transverse sections from two littermate rat pups that were either subjected to a series of binocular TTX injections (Fig. 9A) or subjected to bilateral eye removal on PND 3 (Fig. 9B). Note in both cases

Fig. 7. Photomicrographs comparing the pattern of AChE staining in occipital cortex contralateral to unilateral TI'X injections of an eye (A) and contralateral to a unilateral enucleation (B). Arrowheads indicate the borders of cortical area 17. Bar in A = 500/~m.

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Fig. 8. Densitometric data showing effects on AChE reaction product density profiles in cortical area 17 contralateral to unilateral eye manipulation. A: lateral area 17 contralateral to TTX. B: medial area 17 contralateral to TTX. C: difference between lateral and medial portions of area 17 contralateral to q"rx injections. D: lateral area 17 contralateral to enucleation. E: medial area 17 contralateral to enucleation. F: difference between lateral and medial portions of area 17 contralateral to enucleation.

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Fig. 9. Photomicrographs comparing the pattern of AChE staining in occipital cortex following bilateral T r x injections (A) and bilateral enucleation (B). Arrowheads indicate the borders of cortical area 17. Bar in B - 500 urn. the relative reduction in A C h E reaction product in layer IV of cortical area 17. This decrement includes both the medial and lateral portions of both hemispheres, although light staining remains in lateral portions. C o m p a r e these photomicrographs with those from the normal animal presented in Fig. 1A. While the density levels of A C h E in this case admittedly were low, K o l m o g o r o v - S m i r n o v tests on densitometric data detected no differences in A C h E density profiles between lateral and medial regions of area 17 of one hemisphere, or between the two hemispheres for either the T T X injected or enucleated cases. It is important to note here that the pattern of A C h E reaction product in cortical areas outside primary visual cortex, particularly the primary somatosensory and auditory cortical areas 43 remains unchanged.

Evidence for anterograde transport of HRP by retinal ganglion cells in animals with eye injections of TTX Fig. 10 presents photomicrographs from a rat pup that was subjected to a series of monocular T T X injections on P N D 3, 5, 7, and 9. On P N D 11, this animal was given an injection of a mixture of T T X and

Fig. 10. Photomicrographs showing results of a case in which HRP was injected along with TTX on PND 11. A: ACHEstained transverse section through occipital cortex showing the decrease in AChE activity in the medial portion of cortical area 17 contralateral to eye injections of TTX. Arrowheads indicate borders of area 17. B: HRP-reacted transverse section through dorsal thalamus of the same hemisphere as shown in A showing transport of HRP from the TTX injected eye. Bar = 500pm. H R P (in the same eye as the other injections). The p h o t o m i c r o g r a p h in Fig. 10A illustrates the pattern of A C h E staining in occipital cortex contralateral to the injected eye. Note the reduction in density of the A C h E reaction product in the medial sector of area 17. This decrease in A C h E staining is similar to other cases shown above and is significant by the K o l m o g o r o v - S m i r n o v test (D = 3.22; P < 0.01). Fig. 10B presents a photomicrograph of an HRPreacted transverse section through the dorsal thalamus contralateral to the injection (and ipsilateral to the section in Fig. 10A). Note the presence of H R P reaction product in the optic tract and lateral geniculate body, indicating the presence of H R P transp o r t e d along the axons of retinal ganglion cells from the T T X injected eye. DISCUSSION

Overview of results Several recent studies from this l a b o r a t o r y have

79 demonstrated that rat cortical area 17, primary visual cortex, displays intense AChE activity transiently during early postnatal development 43-49. In this regard, developing visual cortex is similar to developing primary somatosensory 23-26 and auditory cortical areas 43. Work from this laboratory has also demonstrated that the transiently expressed AChE activity is experimentally manipulable; neonatal eye removal results in an absence of the normally expressed transient AChE activity44'49. Results presented in the present manuscript demonstrate that repeated injections of TTX into the vitreous body of the eye also reduce levels of AChE activity, similar to that produced by eye removal. Tetrodotoxin has been used in a variety of studies as an effective blocker of voltage-dependent sodium channels s'1°'32'59. By blocking sodium channels, TTX effectively eliminates formation and propagation of action potentials along axons. In the present case, injection of TTX into the vitreous body of the eyes of infant animals would be expected to block action potential activity of retinal ganglion cells and their axons, which make up the optic nerve and tract. Theoretically, TTX administration would result in a functional removal of optic tract input to the geniculocortical neurons. However, several technical points must be considered before conclusions can be drawn. Technical considerations The first question to be dealt with concerns whether the TTX effectively produced action potential blockade in the optic nerves of our animals. This is difficult to state with certainty because the pupillary reflex is not well developed in these young animals and we did not undertake electrophysiological experiments to test the activity of the optic nerve. However, our experiments demonstrated that equivalent doses of equal volume and strength TTX resulted in absence of pupillary reactivity in juvenile rats. Further, O'Leary et al. 34 have demonstrated that TTX doses smaller than those employed in this study effectively reduced physiological activity in optic nerve of infant rats. We chose to administer the TTX every other day to reduce the chance of damage to the eye due to the injection procedure. We believe administration every other day to be an effective paradigm because evidence from other investigators s,1°,34,59 demon-

strates that the effect of a single administration of TTX lasts for at least 48 h. Indeed, in our own studies of T'I'X injection into vitreous bodies of juvenile animals, the pupillary reflex was still depressed 48 h after administration. However, because of the absence of independent assessment of efficacy of TTX, we must consider the possibility that our TFX paradigm did not result in chronically reduced optic nerve activity. The next issue to be dealt with concerns the possibility that TTX injections may damage the retina and produce a condition effectively similar to enucleation. Such damage could occur either through cytotoxic effects of the TI'X or mechanical or infectious damage to the eye from the injection procedure. However, two points argue against the possibility that repeated TTX injections killed retinal ganglion cells and thus effectively produced an enucleated animal. First, all animals with TTX injections had grossly normal appearing optic nerves at the time of sacrifice. In contrast, surgical enucleation or ocular evisceration in infant rats leads to a rapid disintegration and disappearance of the optic nerve within a few days44,49. We did not undertake detailed histological analyses of retinal ganglion cells because we believe a more sensitive measure of pathology of these cells would be their ability to transport macromolecules. In this context, compelling evidence is provided by the demonstration that retinal ganglion cells of TI'Xinjected eyes are still capable of anterograde axonal transport of HRP (Fig. 10). We interpret the ability to transport peroxidase by TTX-treated retinal ganglion cells as evidence that the cells are viable and that their axons remain substantially biologically normal. Anterograde transport of HRP to the contralateral lateral geniculate body and reduction of AChE activity in contralateral visual cortex of the same animal provides convincing evidence that loss of AChE activity in visual cortex is not due to retinal damage per se. We must add the caveat, however, that the anterograde transport studies only demonstrated that the optic nerve as a whole was able to transport HRP. It is certainly possible that a considerable number of retinal ganglion cells were atrophic after the series of TTX injections. Further, TTX administration may interfere with some aspects of axonal transport without blocking total transport of HRP 4°. Thus,

80 it must be kept in mind that both the injection procedures and TTX itself may have produced a retinal projection that differs from normal in ways other than simply the absence of sodium channel-mediated action potentials. Interpretation of our results also relies upon aspects of AChE histochemistry. The present data are based upon histochemical demonstration of differences in quantity or activity of AChE between different cortical regions within an animal and within the same cortical region between animals. Although we feel confident that a monotonic relationship exists between amount or activity of AChE and density of the AChE reaction product, we have no basis to assume the relationship is linear. Further, we still know relatively little regarding either the regulation of AChE levels or the metabolism of AChE and thus interpretations must be made cautiously.

Comparison of the effects of TTX and enucleation There is a marked resemblance between the pattern of AChE in visual cortex of TTX-injected animals and the pattern of AChE in visual cortex of enucleated animals. This similarity is found whether the animals being compared had received unilateral or bilateral manipulations. Although the experiments employing HRP transport demonstrated that intraocular injections of TTX were not creating an effective enucleation in the animal, the similar change in the pattern of AChE in visual cortex by the two manipulations may result from the same mechanism. In both groups of animals, physiological activity afferent to the dorsal lateral geniculate nucleus is substantially decreased. In enucleation, afferent elements are completely removed, while TTX injections reduce or eliminate action potentials in the afferent axons. Both manipulations profoundly decrease the level of transient AChE activity in the cortex. The degree of reduction of AChE activity in visual cortex in the present study (whether induced by TTX or enucleation) was less than what we have reported previously as a consequence of neonatal enucleation 4~'49. The much more dramatic loss of AChE resulting from PND 0 enucleations indicates again that transient expression of AChE in visual cortex is dependent on normal afferent activity, and also suggests that expression of transient AChE is sensitive to the duration of this afferent activity. Thus it ap-

pears that normal optic nerve activity from birth to PND 3 is able to initiate the cellular processes that result in histochemical staining for AChE in visual cortex, but this process is retarded by subsequent loss of optic nerve afferent activity. Perhaps if animals were injected with TTX on PND 0, a more dramatic decrease in the level of AChE activity would be observed.

Location of transiently expressed AChE As we have discussed previously43-49 the areal and laminar patterns of transiently expressed AChE within occipital cortex are identical with the areal and laminar distributions of geniculocortical axon terminals. This close correspondence suggests that the transiently expressed AChE is associated with these geniculocortical axon terminals. This suggestion receives further support from recent studies 45 demonstrating that placement of lesions in the dLGN result in loss of transiently expressed AChE activity in visual cortex ipsilateral to the lesion. Further, electron microscopic studies currently underway in this laboratory 4s demonstrate that AChE reaction product is associated with granular endoplasmic reticulum of dLGN neurons and with axon terminals in visual cortex of developing rat pups. Thus, although ACHEpositive axons from cell bodies in the basal forebrain are also found in occipital cortex 7'12'25'3°'53, the transient AChE activity appears associated with geniculocortical axons. In summary, current evidence indicates that transiently expressed AChE is produced by protein synthetic machinery of dLGN neurons and transported to visual cortex by geniculocortical axons. In this light, it appears that TTX administration, and the consequent blockage of optic nerve activity, probably interferes with the production or transport of AChE by dLGN neurons. Other recent work from this laboratory supports the suggestion that interfering with retinal projections (either by TTX administration or by enucleation) has its primary effect at the level of the dLGN. That is, we have demonstrated that neonatal enucleation results in a variety of changes in neurons in the dLGN, including loss of neurons, a shrinkage of the remaining dLGN neurons, and a decrease in AChE synthesis by remaining individual dLGN neurons 47.

81

Relationships between visual deprivation, transient expression of ACHE, and development of geniculocortical connectivity A variety of studies over the past several years have demonstrated that visual deprivation can have a profound impact on morphological and physiological aspects of geniculocortical development 13-18'28;~9'33' 38,51,54,55,59-65. One focus of this attention has been directed toward studies of the effect of deprivation on strength of geniculocortical projections. Several aspects of the morphology of geniculocortical projections have been studied, including number and size of dLGN neurons, size of terminal fields of geniculocortical axons, number of dendritic spines of cortical neurons, and size and number of geniculocortical synapses. It would seem reasonable that neonatal visual deprivation would result in a significant decrease in the quantity of geniculocortical projections because enucleation or eyelid suture both have been shown to result in a decreased number of neurons in dLGN 17'19' 33,47,55,62,64. Further, the remaining neurons in dLGN are smaller than neurons from control cases. These decreases in number and size of neurons have been demonstrated in a variety of animals including mice 17, rats 47'62 and primates 17'64. In general, lid suture results in a less dramatic loss of cells than does eye removal. Effects of TTX administration have not been as intensively studied, although Casagrande and Condo 8 recently have shown that repeated intraocular administration of TTX, comparable to the paradigm used in this study, resulted in decreased number and size of neurons in dLGN of the tree shrew. Since visually deprived animals have dLGNs with fewer and smaller neurons, it should not be surprising that the geniculocortical projections from deprived eyes would also be reduced. Animals with significant binocular vision and associated ocular dominance columns provide the best model for examining this issue, and several experiments in cats and primates have demonstrated that ocular dominance columns from deprived eyes are significantly smaller than are columns innervated from normal eyes 17,28,54,59. Ocular dominance columns from layers of the dLGN innervated by the intact eye appear to be expanded and the degree of expansion or compensation appears greater with eye removal than with eyelid suture or

T r x administration. Although comparable data are not available for the rat, the data from other species summarized above suggest that visual deprivation would also result in a decreased geniculocortical projection in the rat. Surprisingly little is known regarding the fate of geniculocortical synapses in visually deprived animals. Early work described relative loss of dendritic spines in visual cortex of enucleated or deprived animals 16'51'52 and while these experiments imply that geniculocortical synapses have been reduced, the evidence is indirect. Several experimenters have used electron microscopic techniques to study number or morphology of synapses in visual cortex during normal development 2-4 and following various forms of visual deprivation 13,14'59-61'65. Many of these experiments have, however, studied synapses that probably are not geniculocortical or synapses of unspecified origins 13,14,65. Tieman 6°,61 however, has used anterograde transport and autoradiographic techniques to study the fate of identified geniculocortical synapses, and reported that geniculocortical synapses are reduced in number and size in eyelid sutured animals. Further, and perhaps most relevant, Riccio and Mathews 41, using a paradigm of TTX injections identical to the paradigm used in the present study, reported that TTX administration resulted in a loss of approximately 20% of asymmetric synapses in layer IV of contralateral visual cortex. These synapses were not identified as to their origin, but were certainly in the right position to be candidates for geniculocortical synapses. Thus, current data indicate that visual deprivation in the rat (whether induced by enucleation or intraocular TTX administration) results in a markedly reduced level of transiently expressed AChE activity in visual cortex. These forms of deprivation also have been shown to reduce geniculocortical terminal fields and numbers of synapses. Whether levels of transiently expressed AChE during development and number of geniculocortical synapses in the mature brain are simply correlated dependent variables or whether number of synapses is dependent upon the transiently expressed AChE 43 remains to be determined.

A developmental rolefor AChE? Perhaps the most interesting aspect of the expres-

82 sion of A C h E in developing animals is its temporal pattern 43. The A C h E is expressed transiently in rat geniculocortical neurons (and in thalamocortical projections to primary somatosensory and auditory cortices as well) during the period of time when their axons are growing into the thalamic recipient layers of cortex and forming synapses with cortical neurons 3"4'23-26'29"43"44"49"50"66. W h e n the period of ingrowth and synaptogenesis has passed, A C h E activity in these thalarnocortical neurons declines markedly. The adult cortex certainly displays A C h E activity 12"19"30"36A253, but the areal and laminar patterns of

43,57, Is transient A C h E expression during thalamocortical axon growth and synaptogenesis simply an e p i p h e n o m e n o n , a by-product of some aspect of the developmental process? Or could A C h E be playing a particular functional role in the formation of geniculocortical connections? Unfortunately we cannot provide an answer at present. It is of particular interest that several reports over the past two decades have described the transient presence of A C h E activity in a variety of regions of the developing central nervous system, particularly during times of synaptogenesis 6'9'11A9"21-27'43-49. Although the issue has been

A C h E activity seen in the developing animal are quite different from those found in the adult. A temporal correlation between the transient expression of A C h E by thalamocortical neurons and time of in-

ACKNOWLEDGEMENTS

growth of these thalamocortical axons into thalamic recipient layers of cortex has also been noted for other thalamocortical projections in primates 21'22. The function, if any, that A C h E may be serving in this developmental process is still unknown 9"1127'

We thank Drs. E . G . Jones, S.H.C. H e n d r y , R.L. M e y e r and D . D . M . O ' L e a r y for stimulating discussions. Supported in part by N S F G r a n t 87-08515 to R . T . R .

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